Soluble lymphotoxin-β receptors and anti-lymphotoxin receptor and ligand antibodies as therapeutic agents for the treatment of immunological disease

ABSTRACT

This invention relates to compositions and methods comprising &#34;lymphotoxin-β receptor blocking agents&#34;, which block lymphotoxin-β receptor signalling. Lymphotoxin-β receptor blocking agents are useful for treating lymphocyte-mediated immunological diseases, and more particularly, for inhibiting Th1 cell-mediated immune responses. This invention relates to soluble forms of the lymphotoxin-β receptor extracellular domain that act as lymphotoxin-β receptor blocking agents. This invention also relates to the use of antibodies directed against either the lymphotoxin-β receptor or its ligand, surface lymphotoxin, that act as lymphotoxin-β receptor blocking agents. A novel screening method for selecting soluble receptors, antibodies and other agents that block LT-β receptor signalling is provided.

TECHNICAL FIELD OF THE INVENTION

This invention relates to compositions and ethods comprising"lymphotoxin-β receptor blocking agents", which block lymphotoxin-βreceptor signalling. lymphotoxin-β receptor blocking agents are usefulfor treating lymphocyte-mediated immunological diseases, and moreparticularly, for inhibiting Th1 cell-mediated immune responses. Thisinvention relates to soluble forms of the lymphotoxin-β receptorextracellular domain that act as lymphotoxin-β receptor blocking agents.This invention also relates to the use of antibodies directed againsteither the lymphotoxin-β receptor or its ligand, surface lymphotoxin,that act as lymphotoxin-β receptor blocking agents. A novel screeningmethod for selecting soluble receptors, antibodies and other agents thatblock LT-β receptor signalling is provided.

BACKGROUND OF THE INVENTION

The pattern of cytokines released at the onset of an immune challengecan affect the subsequent choice of which immune effector pathways areactivated. The choice between immune effector mechanisms is mediated byCD4-positive helper T lymphocytes (T helper cells or Th cells). Th cellsinteract with antigen-presenting cells (APCs), which display peptidefragments of processed foreign antigen in association with MHC class IImolecules on their surfaces. Th cells are activated when they recognizeparticular epitopes of a foreign antigen displayed on the appropriateAPC surface for which the Th cells express a specific receptor.Activated Th cells, in turn, secrete cytokines (lymphokines) whichactivate appropriate immune effector mechanisms.

Th cells can activate diverse effector mechanisms, including killer Tcell activation, B cell antibody production and macrophage activation.The choice between effector mechanisms is mediated largely by whichcytokines are produced by the activated Th cells.

Th cells can be divided into three subgroups based on their cytokinesecretion patterns (Fitch et al., Ann. Rev. Immunol., 11, pp. 29-48(1993)). These subgroups are called Th0, Th1 and Th2. In the mouse,non-stimulated "naive" T helper cells produce IL-2. Short termstimulation leads to Tho precursor cells, which produce a wide range ofcytokines including IFN-γ, IL-2, IL-4, IL-5 and IL-10.Chronically-stimulated Th0 cells can differentiate into either Th1 orTh2 cell types, whereupon the cytokine expression pattern changes.

Some cytokines are released by both Th1 and Th2 cells (e.g., IL-3,GM-CSF and TNF). Other cytokines are made exclusively by one or theother Th cell subgroup. The specialized effects of T helper cellsubgroups were first recognized in mouse. A similar subdivision of Thelper cells also exists in humans (Romagnani et al., Ann. Rev.Immunol., 12 , pp. 227-57 (1994)).

Th1 cells produce LT-α, IL-2 and IFN-γ. In humans, the Th1 pattern ofcytokine secretion has been generally associated with cellular immunityand resistance to infection. The Th1 cytokines tend to activatemacrophages and certain inflammatory responses such as Type IV "delayedtype" hypersensitivity (see below). Th1 cytokines play an important rolein cellular rejection of tissue grafts and organ transplants.

Th2 cells produce the cytokines IL-4, IL-5, IL-6 and IL-10. Th2cytokines increase eosinophil and mast cell production and promote thefull expansion and maturation of B cells (Howard et al., "T cell-derivedcytokines and their receptors", Fundamental Immunology, 3d ed., RavenPress, New York (1993)). Th2 cytokines also enhance antibody production,including IgE antibodies associated with allergic responses andanti-graft antibodies. Th2 cells may also participate in immunesuppression and tolerance to persistent antigens.

Th1- and Th2-associated cytokines play a role in certainhypersensitivity responses--inappropriate or disproportionate immuneresponses evoked upon contact with a previously encountered antigen.There are four recognized types of hypersensitivity (Roitt et al.,Immunology, pp. 19.1-22.12 (Mosby-Year Book Europe Ltd., 3d ed. 1993)).

Type I "immediate hypersensitivity" involves allergen-induced Th2 cellactivation and Th2 cytokine release. The Th2 cytokine IL-4 stimulates Bcells to undergo isotype switching to produce IgE, which activates mastcells to produce acute inflammatory reactions such as those which leadto eczema, asthma and rhinitis.

Types II and III hypersensitivity are caused by IgG and IgM antibodiesdirected against cell surface or specific tissue antigens (Type II) orsoluble serum antigens (Type III). These types of hypersensitivityreactions are not thought to be mediated by Th cells.

Type IV "delayed type" hypersensitivity (DTH) is Th1 cell mediated. DTHreactions take more than 12 hours to develop and are referred to as"cell-mediated" because they can be transferred between mice bytransferring Th1 cells but not serum alone. Type IV DTH responses aregenerally classified into three types: contact, tuberculin-type andgranulomatous hypersensitivity.

Many cell-mediated responses that can cause disease are inducible inhealthy mice by transferring lymphocytes from a diseased mouse (e.g.,insulin-dependent diabetes and experimental autoimmune encephalitis).This feature distinguishes Type IV DTH from the other three types ofhypersensitivity, which are humoral immune responses caused primarily byantibodies which can be transferred in cell-free serum.

T helper cells also participate in the regulation of de novoimmunoglobulin isotype switching. Different Th subsets may influence therelative proportion of immunoglobulins of a given isotype produced inresponse to immune challenge. For example, the Th2 cytokine IL-4 canswitch activated B cells to the IgG1 isotype and suppress otherisotypes. As discussed above, IL-4 also activates IgE overproduction intype I hypersensitivity reactions. The Th2 cytokine IL-5 induces the IgAisotype. These Th2 cytokine effects on isotype switching arecounter-balanced by IFN-γ produced by Th1 cells.

The differential patterns of cytokines secreted by Th1 and Th2 cellsappear to direct a response towards different immune effectormechanisms. The switch that activates either a cell-mediated or humoraleffector mechanism is sensitized by cross-suppression between Th1 andTh2 cells: IFN-γ produced by Th1 cells inhibits Th2 cell proliferationand Th2 cell-secreted IL-10 appears to reduce cytokine secretion fromTh1 cells.

Depending on the relative affinities of the cytokines for theirmolecular targets, the Th1 and Th2 negative regulatory circuits mayamplify the effects of small concentration differences between Th1 andTh2 cytokines. An amplified Th1 or Th2 cytokine signal may trigger theswitch between cell-mediated or humoral effector mechanisms based onsmall changes in the relative concentrations of Th1 and Th2 cytokines.The ability to control this switch by modulating the relativeconcentrations of Th1 and Th2 cytokines would be useful for treatingimbalances in a variety of Th1 and Th2 cell-dependent immune responseswhich can lead to immune disorders and diseases.

Pathological Th1 responses are associated with a number oforgan-specific and systemic autoimmune conditions, chronic inflammatorydiseases, and delayed type hypersensitivity reactions. As discussedabove, Th1 responses also contribute to cellular responses leading tografted tissue and transplanted organ rejection. The treatment of thesevarious Th1 cell-based immunological conditions generally employsimmunomodulatory and immuno suppressive agents as well as a number ofdrugs with poorly characterized mechanisms (e.g., gold orpenicillamine). Three general immunosuppressive agents used currentlyare steroids, cyclosporine and azathioprine.

Steroids are pleiotropic anti-inflammatory agents which suppressactivated macrophages and inhibit the activity of antigen presentingcells in ways which reverse many of the effects of the Th1 cytokineIFN-γ. Cyclosporine--a potent immunosuppressive agent--suppressescytokine production and reduces the expression of IL-2 receptors onlymphocytes during their activation. Azathioprine is ananti-proliferative agent which inhibits DNA synthesis. Thesenon-specific immunosuppressive agents are generally required in highdoses which increase their toxicity (e.g. nephro- and hepatotoxicity)and cause adverse side effects. They are thus unsuitable for long termtherapies.

To address the problems caused by conventional treatments withnon-specific immunosuppressive agents, many current therapeuticstrategies aim at suppressing or activating selective aspects of theimmune system. An especially attractive goal is the manipulation of thebalance between Th1 and Th2 cytokines to shift the balance betweencell-mediated and humoral effector mechanisms.

To accomplish a shift between cell-mediated and humoral effectormechanisms, it would be useful to be able to modulate the activity of amolecule that can shift the relative activities of Th1 and Th2 cellsubclasses. Candidates for such molecules include the cytokines andtheir receptors. Recent data suggest that LT-α, IL-12, IFN-α and IFN-γfavor the development of Th1 responses, whereas IL-1 and IL-4 polarize aresponse towards a Th2 effector mechanism (Romagnani et al., Ann. Rev.Immunol., 12 , pp. 227-57 (1994)).

Many of the Th cell cytokines are pleiotropic regulators of immunedevelopment and function, and inhibiting their production would havedeleterious effects on non-T cell mediated responses. A desirable andeffective target for selectively modulating the choice between Th1 andTh2 effector mechanisms has not been identified.

SUMMARY OF THE INVENTION

The present invention solves the problems referred to above by providingpharmaceutical compositions and methods for treating immunologicaldiseases by inhibiting lymphotoxin-β receptor (LT-β-R) signalling usinglymphotoxin-β receptor blocking agents. More particularly, thecompositions and methods comprising LT-β-R blocking agents are usefulfor inhibiting Th1 cell-mediated immune responses.

In one embodiment, soluble forms of the lymphotoxin-β receptorextracellular domain that act as LT-β-R blocking agents are provided.The preferred compositions and methods of this embodiment comprise arecombinant lymphotoxin-β receptor fusion protein that has the LT-β-Rextracellular ligand binding domain fused to an immunoglobulin constantheavy chain domain. More preferably, the LT-β-R ligand binding domain isfused to a human IgG Fc domain.

In another embodiment of this invention, antibodies that act as LT-β-Rblocking agents are provided. Preferred compositions and methods of thisembodiment comprise one or more antibodies directed against thelymphotoxin-β receptor. More preferably, the antibody is a monoclonalantibody. Other preferred compositions and methods of this embodimentcomprise one or more antibodies directed against surface lymphotoxin.More preferably, the antibody is a monoclonal antibody directed againstlymphotoxin-β.

This invention further provides a novel screening process for selectingLT-β-R blocking agents----such as soluble forms of the LT-β-R, anti-LTAbs and anti-LT-β-R Abs. This screening process involves performingtumor cell cytotoxicity assays that monitor LT-β-R signalling. The assaymakes use of the increased sensitivity of human adenocarcinoma cells toligand- or antibody-induced LT-β-R signalling in the presence of anLT-β-R activating agent (such as LT-α1/β2) in a tumor cytotoxicityassay.

LT-β-R blocking agents inhibit the cytotoxic effects of LT-α/βheteromeric complexes (or other LT-β-R activating agents) on tumorcells. The procedure used to test putative LT-β-R blocking agents isexemplified for the case of anti-LT-β-R antibodies (in the presence ofthe LT-β-R activating agents LT-α1/β2) and comprises the followingsteps:

1) Tumor cells (e.g., HT29 human adenocarcinoma cells) are cultured forseveral days in media containing IFN-γ and purified LT-α1/β2 in thepresence or absence of the particular anti-LT-β-R Ab being assayed;

2) The cells are treated with a dye that stains living cells; and

3) The number of stained cells is quantitated to determine the fractionof tumor cells killed in the presence of LT-α1/β2, IFN-γ and the testanti-LT-β-R Ab in each sample. Alternatively, the number of survivingcells can be determined by any of a number of well-known assays whichmeasure cell viability, such as ³ H-thymidine incorporation into DNA. Ananti-LT-β-R Ab (or an Ab combination) that decreases the percentage oftumor cells killed in this assay by at least 20% is a LT-β-R blockingagent within the scope of this invention.

This cytolytic assay may be performed using LT-α/β heteromeric complexesand other LT-β-R activating agents, either alone or in combination. Theassay can also be adapted as required to identify new LT-β-R blockingagents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The sequence of the extracellular portion of the human LT-βreceptor which encodes the ligand binding domain (SEQ ID NO:1).

FIG. 2. A soluble murine LT-β receptor coupled to the human IgG1 Fcdomain (mLT-β-R-Fc) blocks LT-β-R signalling in mouse WEHI 164 cellsinduced by soluble murine LT-α/β ligand. WEHI 164 cells are killed as afunction of increasing LT ligand (mLT-α/β) concentration. SolublemLT-β-R-Fc (10 μg/ml) blocks this LT ligand-induced cell death. Asoluble murine TNF receptor fusion protein (p55TNF-R-Fc) has littleeffect on blocking LT-α/β-activated cell death. Growth was quantitatedafter three days by measuring the optical density (OD 550) of reactedMTT, which is proportional to cell number.

FIG. 3. An antibody directed against human LT-β-R (BDA8 mAb) blocks theinteraction between soluble LT ligand and LT-β-R on a human cellsurface. The growth of WiDr tumor cells is blocked by a combination ofIFN-γ and soluble LT-α1/β2 ligand. The anti-LT-β-R antibody BDA8 blocksthe ability of LT-α1/β2 ligand to inhibit the growth of WiDr tumorcells. Solid symbols show cell growth in the presence of IgGI controlmAb (10 μg/ml). Open symbols show the effects of anti-LT-β-R mAb BDA8(10 μg/ml).

FIG. 4. An antibody directed against human LT-β (B9 mAb) blocks theinteraction between cell surface LT-α/β ligand and soluble LT-β receptor(hLT-β-R-Fc; 2μg/ml). Surface bound LT-β-R-Fc was detected usingphycoerythrin-labelled donkey anti-human IgG and FACS analysis. The meanfluorescence intensity of the resultant peak is plotted as channelnumber. Dotted line shows the mean fluorescence intensity correspondingto the amount of receptor bound in the absence of the B9 mAb.

FIG. 5. The effects of a LT-β-R blocking agent (mLT-β-R-Fc) on earswelling in a mouse contact delayed type hypersensitivity model (DTH).The graph shows the increase in ear thickness measured 24 hoursfollowing 0.2% DNFB antigen challenge onto the ears of sensitized mice.Each symbol represents a separate experiment. All experiments utilized7-8 animals per point except those demarcated with a diamond, which usedonly 4 animals per point. Mice treated with buffer (PBS) and with 20mg/kg of a control IgG fusion protein (LFA3-Fc) served as negativecontrols. Mice treated with 8 mg/kg of an anti-VLA4 mAb (PS/2 mAb),which inhibits contact DTH ear swelling, served as positive controls.

FIG. 6A-C. Treatment with a soluble LT-β-R receptor in vivo does notalter ovalbumin-specific antibody titers following primary immunizationof mice with ovalbumin (a Th2 type response). Mice treated with eithernothing, 100-200 μg of a LT-β-R fusion protein (mLT-β-R-Fc) or of acontrol fusion protein (human LFA3-Fc) were immunized with 100 μgovalbumin in complete Freund's adjuvant at the tail base. Eleven daysafter the immunization, the mice were sacrificed and the serumanti-ovalbumin antibody titers determined by performing isotype-specificELISAs. Each point represents the inverse of the serum titer from anindividual animal. Data from three separate experiments are indicatedwith each symbol corresponding to a common experiment produced by thehybridoma cell line BD.A8.AB9 (ATCC Accession No: HB11798).

FIG. 7. Treatment with a soluble LT-β receptor in vivo does not affectprimed lymphocyte response to restimulation with an antigen (a Th2 typeresponse). Mice treated with either nothing, 200 μg of a LT-β-R fusionprotein (mLT-β-R-Fc) or 200 μg of a control fusion protein (humanLFA3-Fc) were immunized with 100 μg of ovalbumin in complete Freund'sadjuvant at the tail base. Eleven days after the immunization, the micewere sacrificed and lymph node cells were isolated, cultured, andrestimulated with ovalbumin antigen. Proliferation of stimulated cellswas assessed by measuring ³ H-thymidine incorporation. Open squares showthe levels of ³ H-thymidine incorporated by ovalbumin-stimulated lymphnode cells from mLT-β-R-Fc-treated mice. Solid squares represent ³H-thymidine incorporation by lymph node cells from mice treated with acontrol IgG fusion protein (LFA3-Fc). Each line represents cells fromone mouse.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention herein described may be fully understood,the following detailed description is set forth.

The term "cytokine" refers to a molecule which mediates interactionsbetween cells. A "lymphokine" is a cytokine released by lymphocytes.

The term "T helper (Th) cells" refers to a functional subclass of Tcells which help to generate cytotoxic T cells and which cooperate withB cells to stimulate antibody production. Helper T cells recognizeantigen in association with class II MHC molecules.

The term "Th1" refers to a subclass of T helper cells that produce LT-α,interferon-γ and IL-2 (and other cytokines) and which elicitinflammatory reactions associated with a cellular, i.e.non-immunoglobulin, response to a challenge.

The term "Th2"refers to a subclass of T helper cells that producecytokines, including IL-4, IL-5, IL-6 and IL-10, which are associatedwith an immunoglobulin (humoral) response to an immune challenge.

The term "cell mediated" refers to those immunological events thatresult from the direct effects of T cells and their products to producea response. This type of response is generally (but not exclusively)associated with the Th1 class of T cells. Not included in this categorywould be the helper effects of T cells on B cell differentiation and Bcell expansion, which are generally associated with the Th2 class of Tcells.

The term "delayed type hypersensitivity (DTH)" refers to animmunological response that is characterized by a slow response to anantigen with the full effect manifesting itself over a 1-3 day period.This slow response is in contrast to the relatively fast response seenin an immunoglobulin-mediated (humoral) allergic reaction. There arethree types of DTH reactions: contact hypersensitivity, tuberculin-typehypersensitivity and granulomatous reactions.

The terms "immunoglobulin response" or "humoral response" refer to theimmunological response of an animal to a foreign antigen whereby theanimal produces antibodies to the foreign antigen. The Th2 class of Thelper cells are critical to the efficient production of high affinityantibodies.

The term "Fc domain" of an antibody refers to a part of the moleculecomprising the hinge, CH2 and CH3 domains, but lacking the antigenbinding sites. The term is also meant to include the equivalent regionsof an IgM or other antibody isotype.

The term "anti-LT-β receptor antibody" refers to any antibody thatspecifically binds to at least one epitope of the LT-β receptor.

The term "anti-LT antibody" refers to any antibody that specificallybinds to at least one epitope of LT-α, LT-β or a LT-α/β complex.

The term "LT-β-R signalling" refers to molecular reactions associatedwith the LT-β-R pathway and subsequent molecular reactions which resulttherefrom.

The term "LT-β-R blocking agent" refers to an agent that can diminishligand binding to LT-β-R, cell surface LT-β-R clustering or LT-β-Rsignalling, or that can influence how the LT-β-R signal is interpretedwithin the cell.

A LT-β-R blocking agent that acts at the step of ligand-receptor bindingcan inhibit LT ligand binding to the LT-β-R by at least 20%. A LT-β-Rblocking agent that acts after the step of ligand-receptor binding caninhibit the cytotoxic effects of LT-β-R activation on a tumor cell by atleast 20%. Examples of LT-β-R blocking agents include soluble LT-β-R-Fcmolecules, and anti-LT-α, anti-LT-β, anti-LT-α/β and anti-LT-β-R Abs.Preferably, the antibodies do not cross-react with the secreted form ofLT-α.

The term "LT-β-R biological activity" refers to: 1) the ability of theLT-β-R molecule or derivative to compete for soluble or surface LTligand binding with soluble or surface LT-β-R molecules; or 2) theability to stimulate an immune regulatory response or cytotoxic activityin common with a native LT-β-R molecule.

The terms "LT-α/β heteromeric complex" and "LT heteromeric complex"refer to a stable association between at least one LT-α and one or moreLT-β subunits, including soluble, mutant, altered and chimeric forms ofone or more of the subunits. The subunits can associate throughelectrostatic, van der Waals, or covalent interactions. Preferably, theLT-α/62 heteromeric complex has at least two adjacent LT-β subunits andlacks adjacent LT-α subunits. When the LT-α/β heteromeric complex servesas a LT-β-R activating agent in a cell growth assay, the complex ispreferably soluble and has the stoichiometry LT-α1/β2.

Soluble LT-α/62 heteromeric complexes lack a transmembrane domain andcan be secreted by an appropriate host cell which has been engineered toexpress LT-α and/or LT-β subunits (Crowe et al., J. Immunol. Methods,168, pp. 79-89 (1994)).

The term "LT ligand" refers to a LT heteromeric complex or derivativethereof that can specifically bind to the LT-β receptor.

The term "LT-β-R ligand binding domain" refers to the portion orportions of the LT-β-R that are involved in specific recognition of andinteraction with a LT ligand.

The terms "surface LT-α/62 complex" and "surface LT complex" refer to acomplex comprising LT-α and membrane-bound LT-β subunits--includingmutant, altered and chimeric forms of one or more of the subunits--whichis displayed on the cell surface. "Surface LT ligand" refers to asurface LT complex or derivative thereof that can specifically bind tothe LT-β receptor.

The term "subject" refers to an animal, or to one or more cells derivedfrom an animal. Preferably, the animal is a mammal. Cells may be in anyform, including but not limited to cells retained in tissue, cellclusters, immortalized, transfected or transformed cells, and cellsderived from an animal that have been physically or phenotypicallyaltered.

Lymphotoxin-β: A Member of the TNF Family

Tumor Necrosis Factor (TNF)-related cytokines have emerged as a largefamily of pleiotropic mediators of host defense and immune regulation.Members of this family exist in membrane-bound forms which act locallythrough cell-cell contact, or as secreted proteins which can act ondistant targets. A parallel family of TNF-related receptors react withthese cytokines and trigger a variety of pathways including cell death,cell proliferation, tissue differentiation and proinflammatoryresponses.

TNF, lymphotoxin-α (LT-α, also called TNF-β) and lymphotoxin-β (LT-β)are members of the TNF family of ligands, which also includes theligands to the Fas, CD27, CD30, CD40, OX-40 and 4-1BB receptors (Smithet al., Cell, 76, pp. 959-62 (1994)). Signalling by several members ofthe TNF family--including TNF, LT-α, LT-β and Fas--can induce tumor celldeath by necrosis or apoptosis (programmed cell death). Innon-tumorigenic cells, TNF and many of the TNF family ligand-receptorinteractions influence immune system development and responses tovarious immune challenges.

Most TNF family ligands are found as a membrane-bound form on the cellsurface. TNF and LT-α are found in both secreted and membrane-associatedsurface forms in humans. Surface TNF has a transmembrane region that isproteolytically cleaved to generate the secreted form. In contrast,surface LT-α lacks a transmembrane region. Membrane-associated LT-α istethered to the cell surface as a heteromeric complex with LT-β, arelated transmembrane polypeptide, in a LT-α/62 complex.

Most membrane-associated LT-α/62 complexes ("surface LT") have aLT-α1/β2 stoichiometry (Browning et al., Cell, 72, pp. 847-56 (1993);Browning et al., J. Immunol., 154, pp. 33-46 (1995)). Surface LT ligandsdo not bind TNF-R with high affinity and do not activate TNF-Rsignalling. Another TNF-related receptor, called the LT-β receptor(LT-β-R), binds these surface lymphotoxin complexes with high affinity(Crowe et al., Science, 264, pp. 707-10 (1994)).

LT-β-R signalling, like TNF-R signalling, has anti-proliferative effectsand can be cytotoxic to tumor cells. In applicants' co-pending U.S.application Ser. No. 08/378,968, compositions and methods forselectively stimulating LT-β-R using LT-β-R activating agents aredisclosed. LT-β-R activating agents are useful for inhibiting tumor cellgrowth without co-activating TNF-R-induced proinflammatory orimmunoregulatory pathways.

In non-tumor cells, TNF and TNF-related cytokines are active in a widevariety of immune responses. Both TNF and LT-α ligands bind to andactivate TNF receptors (p55 or p60 and p75 or p80; herein called"TNF-R"). TNF and LT-α are produced by macrophages in an early and rapidresponse to microbial infection which enhances the microbiocidalactivity of macrophages and neutrophils. TNF and LT-α made bymacrophages or cytotoxic T lymphocytes (CTLs or "killer T cells") bindto TNF receptors on target cell surfaces and trigger the death ofsusceptible cells.

TNF and TNF-related cytokines can also initiate inflammatory cascades inresponse to infection or stress. The release of TNF, LT-α and IFN-γchanges the adhesion properties between the vascular endothelial cellsand certain lymphocyte types. Increased adhesion facilitates phagocyteand leucocyte migration from the bloodstream into the tissuessurrounding an inflammation site. Similar inflammatory reactions play amajor role in cellular rejection of tissue grafts and organ transplants,and in certain immune disorders.

Cell surface lymphotoxin (LT) complexes have been characterized in CD4⁺T cell hybridoma cells (II-23.D7) which express high levels of LT(Browning et al., J. Immunol., 147, pp. 1230-37 (1991); Androlewicz etal., J. Biol. Chem., 267, pp. 2542-47 (1992)). The expression andbiological roles of LT-β-R, LT subunits and surface LT complexes havebeen reviewed in C. F. Ware et al., "The ligands and receptors of thelymphotoxin system", in Pathways for Cytolysis, Current TopicsMicrobiol. Immunol., Springer-Verlag, pp. 175-218 (1995).

LT-α expression is induced and LT-α secreted primarily by activated Tand B lymphocytes and natural killer (NK) cells. Among the T helper cellsubclasses, LT-α appears to be produced by Th1 but not Th2 cells. LT-αhas also been detected in melanocytes. Microglia and T cells in lesionsof multiple sclerosis patients can also be stained with anti-LT-αantisera.

Lymphotoxin-β (also called p33), has been identified on the surface of Tlymphocytes, T cell lines, B cell lines and lymphokine-activated killer(LAK) cells. LT-β is the subject of applicants' co-pending internationalapplications PCT/US91/04588, published Jan. 9, 1992 as WO 92/00329; andPCT/US93/11669, published Jun. 23, 1994 as WO 94/13808, which are hereinincorporated by reference.

Surface LT complexes are primarily expressed by activated T and Blymphocytes and natural killer (NK) cells as defined by FACS analysis orimmunohistology using anti-LT-α antibodies or soluble LT-β-R-Fc fusionproteins. Surface LT has also been described on human cytotoxic Tlymphocyte (CTL) clones, activated peripheral mononuclear lymphocytes(PML), IL-2-activated peripheral blood lymphocytes (LAK cells), pokeweedmitogen-activated or anti-CD40-activated peripheral B lymphocytes (PBL)and various lymphoid tumors of T and B cell lineage. Engagement ofalloantigen-bearing target cells specifically induces surface LTexpression by CD8⁺ and CD4⁺ CTL clones.

The LT-β receptor, a member of the TNF family of receptors, specificallybinds to surface LT ligands. LT-β-R binds LT heteromeric complexes(predominantly LT-α1/β2 and LT-α2/β1) but does not bind TNF or LT-α(Crowe et al., Science, 264, pp. 707-10 (1994)). Signalling by LT-β-Rmay play a role in peripheral lymphoid organ development and in humoralimmune responses.

Studies on LT-β-R expression are in their early stages. LT-β-R mRNAs arefound in human spleen, thymus and other major organs. LT-β-R expressionpatterns are similar to those reported for p55-TNF-R except that LT-β-Ris lacking on peripheral blood T cells and T cell lines.

Production of soluble LT complexes

Soluble LT-α/β heteromeric complexes comprise LT-β subunits which havebeen changed from a membrane-bound to a soluble form. These complexesare described in detail in applicants' co-pending internationalapplication (PCT/US93/11669, published Jun. 23, 1994 as WO 94/13808).Soluble LT-β peptides are defined by the amino acid sequence oflymphotoxin-β wherein the sequence is cleaved at any point between theend of the transmembrane region (i.e. at about amino acid #44) and thefirst TNF homology region (i.e. at amino acid #88) according to thenumbering system of Browning et al., Cell, 72, pp. 847-56 (1993).

Soluble LT-β polypeptides may be produced by truncating the N-terminusof LT-β to remove the cytoplasmic tail and transmembrane region (Croweet al., Science, 264, pp. 707-710 (1994)). Alternatively, thetransmembrane domain may be inactivated by deletion, or by substitutionof the normally hydrophobic amino acid residues which comprise atransmembrane domain with hydrophilic ones. In either case, asubstantially hydrophilic hydropathy profile is created which willreduce lipid affinity and improve aqueous solubility. Deletion of thetransmembrane domain is preferred over substitution with hydrophilicamino acid residues because it avoids introducing potentiallyimmunogenic epitopes.

The deleted or inactivated transmembrane domain may be replaced with orattached to a type I leader sequence (e.g. the VCAM-1 leader) such thatthe protein is secreted beginning with a sequence anywhere from betweenval40 to pro88. Soluble LT-β polypeptides may include any number ofwell-known leader sequences at the N-terminus. Such a sequence wouldallow the peptides to be expressed and targeted to the secretion pathwayin a eukaryotic system. See, e.g., Ernst et al., U.S. Pat. No. 5,082,783(1992).

Soluble LT-α/β heteromeric complexes may be produced by co-transfectinga suitable host cell with DNA encoding LT-α and soluble LT-β (Crowe etal., J. Immunol. Methods, 168, pp. 79-89 (1994)). Soluble LT-β secretedin the absence of LT-α is highly oligomerized. However, whenco-expressed with LT-α, a 70 kDa trimeric-like structure is formed whichcontains both proteins. It is also possible to produce soluble LT-α 1/β2heteromeric complexes by transfecting a cell line which normallyexpresses only LT-α (such as the RPMI 1788 cells discussed above) with agene encoding a soluble LT-β polypeptide.

LT-α and LT-β polypeptides may be separately synthesized, denaturedusing mild detergents, mixed together and renatured by removing thedetergent to form mixed LT heteromeric complexes which can be separated(see below).

Purification of LT-α 1/β2 Complexes

Soluble LT-α1/β2 heteromeric complexes are separated from co-expressioncomplexes comprising a different subunit stoichiometry by chromatographyusing TNF and LT-β receptors as affinity purification reagents. The TNFreceptors only bind within α/α clefts of LT complexes. The LT-β receptorbinds with high affinity to β/β clefts, and with lower affinity to α/βclefts of heteromeric LT-α/β complexes.

Accordingly, LT-α3 and LT-α2/β1 will bind to TNF-R. The LT-β-R can alsobind LT-α2/β1 trimers (within the α/β clefts) but cannot bind LT-α3. Inaddition, the LT-β-R (but not TNF-R) binds LT-α1/β2 and LT-βn (the exactcomposition of such preparation is unknown, however, they are largeaggregates).

The receptor affinity reagents can be prepared as either a solubleextracellular domain (see for example Loetscher et al., J. Biol. Chem.,266, pp. 18324-29 (1991)), or as chimeric proteins with theextracellular ligand binding domain coupled to an immunoglobulin Fcdomain (Loetscher et al., J. Biol. Chem., 266, pp. 18324-29 (1991);Crowe et al., Science, 264, pp. 707-710 (1994)). Receptors are coupledto affinity matrices by chemical cross-linking using routine procedures.

There are two schemes by which the LT-α1/β2 ligand can be purified usingreceptors and immuno-affinity chromatography. In the first scheme, asupernatant from an appropriate expression system co-expressing bothLT-α and the truncated LT-β form is passed over a TNF-R column. TheTNF-R will bind LT-α3 and LT-α2/β1 trimers. The flow through from theTNF-R column will contain LT-β(n) and LT-α1/β2.

In the second scheme, all LT-β-containing forms (LT-β(n), LT-α1/β2 andLT-α2/β1) are bound to and eluted from a LT-β-R column using classicalmethods such as chaotrophe or pH change. (LT-α3 flows through thiscolumn). The eluate is neutralized or the chaotrophe removed, and theeluate is then passed over a TNF-R column, which binds only to theLT-α2/β1 trimers. The flow through of this column will contain LT-β(n)and LT-α1/β2 trimers.

In both cases, pure LT-α1/β2 trimers can be separated from LT-β bysubsequent gel filtration and/or ion exchange chromatographic proceduresknown to the art.

Alternatively, different forms of LT-α/β heteromeric complexes can beseparated and purified by a variety of conventional chromatographicmeans. It may also be preferable to combine a series of conventionalpurification schemes with one of the immunoaffinity purification stepsdescribed above.

Screening For LT-β-R Blocking Agents

In one embodiment of this invention, the LT-β-R blocking agent comprisesan antibody (Ab) directed against LT-β-R that inhibits LT-β-Rsignalling. Preferably, the anti-LT-β-R Ab is a monoclonal antibody(mAb). One such inhibitory anti-LT-β-R mAb is BDA8 mAb.

Inhibitory anti-LT-β-R Abs and other LT-β-R blocking agents can beidentified using screening methods that detect the ability of one ormore agents either to bind to the LT-β-R or LT ligand, or to inhibit theeffects of LT-β-R signalling on cells.

One screening method makes use of the cytotoxic effects of LT-β-Rsignalling on tumor cells bearing the LT-β-R. Tumor cells are exposed toone or more LT-β-R activating agents to induce LT-β-R signalling. LT-β-Ractivating agents include LT-α/62 heteromeric complexes (preferablysoluble LT-α1/β2) in the presence of IFN-γ, or an activating anti-LT-β-RAb (see below; also described in applicants' co-pending U.S. applicationSer. No. 08/378,968).

Antibodies and other agents that can block LT-β-R signalling areselected based on their ability to inhibit the cytotoxic effect ofLT-β-R signalling on tumor cells in the following assay:

1) Tumor cells such as HT29 cells are cultured for three to four days ina series of tissue culture wells containing media and at least oneLT-β-R activating agent in the presence or absence of serial dilutionsof the agent being tested;

2) A vital dye stain which measures mitochondrial function such as MTTis added to the tumor cell mixture and reacted for several hours;

3) The optical density of the mixture in each well is quantitated at 550nm wavelength light (OD 550). The OD 550 is proportional to the numberof tumor cells remaining in the presence of the LT-β-R activating agentand the test LT-β-R blocking agent in each well. An agent or combinationof agents that can reduce LT-β-R-activated tumor cell cytotoxicity by atleast 20% in this assay is a LT-β-R blocking agent within the scope ofthis invention.

Any agent or combination of agents that activate LT-β-R signalling canbe used in the above assay to identify LT-β-R blocking agents. LT-β-Ractivating agents that induce LT-β-R signalling (such as activatinganti-LT-β-R mAbs) can be selected based on their ability--alone or incombination with other agents--to potentiate tumor cell cytotoxicityusing the tumor cell assay described above.

Another method for selecting an LT-β-R blocking agent is to monitor theability of the putative agent to directly interfere with LTligand-receptor binding. An agent or combination of agents that canblock ligand-receptor binding by at least 20% is an LT-β-R blockingagent within the scope of this invention.

Any of a number of assays that measure the strength of ligand-receptorbinding can be used to perform competition assays with putative LT-β-Rblocking agents. The strength of the binding between a receptor andligand can be measured using an enzyme-linked immunoadsorption assay(ELISA) or a radio-immunoassay (RIA). Specific binding may also bemeasured by fluorescently labelling antibody-antigen complexes andperforming fluorescence-activated cell sorting (FACS) analysis, or byperforming other such immunodetection methods, all of which aretechniques well known in the art.

The ligand-receptor binding interaction may also be measured with theBIAcore™ instrument (Pharmacia Biosensor) which exploits plasmonresonance detection (Zhou et al., Biochemistry, 32, pp. 8193-98 (1993);Faegerstram and O'Shannessy, "Surface plasmon resonance detection inaffinity technologies", in Handbook of Affinity Chromatography, pp.229-52, Marcel Dekker, Inc., New York (1993)).

The BIAcore™ technology allows one to bind receptor to a gold surfaceand to flow ligand over it. Plasmon resonance detection gives directquantitation of the amount of mass bound to the surface in real time.This technique yields both on and off rate constants and thus aligand-receptor dissociation constant and affinity constant can bedirectly determined in the presence and absence of the putative LT-β-Rblocking agent.

With any of these or other techniques for measuring receptor-ligandinteractions, one can evaluate the ability of a LT-β-R blocking agent,alone or in combination with other agents, to inhibit binding of surfaceor soluble LT ligands to surface or soluble LT-β-R molecules. Suchassays may also be used to test LT-β-R blocking agents or derivatives ofsuch agents (e.g. fusions, chimeras, mutants, and chemically alteredforms)--alone or in combination--to optimize the ability of that alteredagent to block LT-β-R activation.

Production of Soluble LT-β-R Molecules

The LT-β-R blocking agents in one embodiment of this invention comprisesoluble LT-β receptor molecules. FIG. 1 shows the sequence of theextracellular portion of the human LT-β-R, which encodes the ligandbinding domain. Using the sequence information in FIG. 1 and recombinantDNA techniques well known in the art, functional fragments encoding theLT-β-R ligand binding domain can be cloned into a vector and expressedin an appropriate host to produce a soluble LT-β-R molecule. SolubleLT-β-R molecules that can compete with native LT-β receptors for LTligand binding according to the assays described herein are selected asLT-β-R blocking agents.

A soluble LT-β receptor comprising amino acid sequences selected fromthose shown in FIG. 1 may be attached to one or more heterologousprotein domains ("fusion domain") to increase the in vivo stability ofthe receptor fusion protein, or to modulate its biological activity orlocalization.

Preferably, stable plasma proteins--which typically have a half-lifegreater than 20 hours in the circulation--are used to construct thereceptor fusion proteins. Such plasma proteins include but are notlimited to: immunoglobulins, serum albumin, lipoproteins,apolipoproteins and transferrin. Sequences that can target the solubleLT-β-R molecule to a particular cell or tissue type may also be attachedto the LT-β-R ligand binding domain to create a specifically-localizedsoluble LT-β-R fusion protein.

All or a functional portion of the LT-β-R extracellular region (FIG. 1)comprising the LT-β-R ligand binding domain may be fused to animmunoglobulin constant region like the Fc domain of a human IgG1 heavychain (Browning et al., J. Immunol., 154, pp. 33-46 (1995)). Solublereceptor-IgG fusion proteins are common immunological reagents andmethods for their construction are known in the art (see e.g., U.S. Pat.No. 5,225,538).

A functional LT-β-R ligand binding domain may be fused to animmunoglobulin (Ig) Fc domain derived from an immunoglobulin class orsubclass other than IgG1. The Fc domains of antibodies belonging todifferent Ig classes or subclasses can activate diverse secondaryeffector functions. Activation occurs when the Fc domain is bound by acognate Fc receptor. Secondary effector functions include the ability toactivate the complement system, to cross the placenta, and to bindvarious microbial proteins. The properties of the different classes andsubclasses of immunoglobulins are described in Roitt et al., Immunology,p. 4.8 (Mosby-Year Book Europe Ltd., 3d ed. 1993).

Activation of the complement system initiates cascades of enzymaticreactions that mediate inflammation. The products of the complementsystem have a variety of functions, including binding of bacteria,endocytosis, phagocytosis, cytotoxicity, free radical production andsolubilization of immune complexes.

The complement enzyme cascade can be activated by the Fc domains ofantigen-bound IgG1 , IgG3 and IgM antibodies. The Fc domain of IgG2appears to be less effective, and the Fc domains of IgG4, IgA, IgD andIgE are ineffective at activating complement. Thus one can select a Fcdomain based on whether its associated secondary effector functions aredesirable for the particular immune response or disease being treatedwith the LT-β-R-Fc fusion protein.

If it would be advantageous to harm or kill the LT ligand-bearing targetcell, one could select an especially active Fc domain (IgG1 ) to makethe LT-β-R-Fc fusion protein. Alternatively, if it would be desirable totarget the LT-β-R-Fc fusion to a cell without triggering the complementsystem, an inactive IgG4 Fc domain could be selected.

Mutations in Fc domains that reduce or eliminate binding to Fc receptorsand complement activation have been described (S. Morrison, Annu. Rev.Immunol., 10, pp. 239-65 (1992)). These or other mutations can be used,alone or in combination, to optimize the activity of the Fc domain usedto construct the LT-β-R-Fc fusion protein.

The production of a soluble human LT-β-R fusion protein comprisingligand binding sequences fused to a human immunoglobulin Fc domain(hLT-β-R-Fc) is described in Example 1. One CHO line made according toExample 1 that secretes hLT-β-R-Fc is called "hLTβ;R-hG1 CHO#14". Asample of this line was deposited on Jul. 21, 1995 with the AmericanType Culture Collection (ATCC) (Rockville, Md.) according to theprovisions of the Budapest Treaty and was assigned the ATCC accessionnumber CRL 11965.

The production of a soluble murine LT-β-R fusion molecule (mLT-β-R-Fc)is described in Example 2. A CHO line made according to Example 2 thatsecretes mLT-β-R-Fc is called "mLTβ;R-hG1 CHO#1.3.BB". A sample of thisline was deposited on Jul. 21, 1995 with the American Type CultureCollection (ATCC) (Rockville, Md.) according to the provisions of theBudapest Treaty and was assigned the ATCC accession number CRL 11964.

All restrictions on the availability to the public of the above ATCCdeposits will be irrevocably removed upon the granting of a patent onthis application.

Different amino acid residues forming the junction point of thereceptor-Ig fusion protein may alter the structure, stability andultimate biological activity of the soluble LT-β receptor fusionprotein. One or more amino acids may be added to the C-terminus of theselected LT-β-R fragment to modify the junction point with the selectedfusion domain.

The N-terminus of the LT-β-R fusion protein may also be varied bychanging the position at which the selected LT-β-R DNA fragment iscleaved at its 5' end for insertion into the recombinant expressionvector. The stability and activity of each LT-β-R fusion protein may betested and optimized using routine experimentation and the assays forselecting LT-β-R blocking agents described herein.

Using the LT-β-R ligand binding domain sequences within theextracellular domain shown in FIG. 1, amino acid sequence variants mayalso be constructed to modify the affinity of the soluble LT-β receptoror fusion protein for LT ligand. The soluble LT-β-R molecules of thisinvention can compete for surface LT ligand binding with endogenous cellsurface LT-β receptors. It is envisioned that any soluble moleculecomprising a LT-β-R ligand binding domain that can compete with cellsurface LT-β receptors for LT ligand binding is a LT-β-R blocking agentthat falls within the scope of the present invention.

Soluble LT-β-R Molecules As LT-β-R Blocking Agents

A soluble human LT-β receptor-immunoglobulin fusion protein (hLT-β-R-Fc)was made according to the procedures in Example 1 and tested for itsability to block LT-β-R-induced cytotoxicity in human HT29 tumor cells.Table 1 (Example 3) compares the ability of soluble LT-β receptor(hLT-β-R-Fc) and TNF receptor (p55-TNF-R-Fc) fusion proteins to blockthe inhibitory effects of various TNF and soluble LT ligands on HT29tumor cell growth.

The data in Table 1 indicate the concentrations at which a soluble LT-βreceptor (hLT-β-R-Fc) can block the tumor cell death caused byinteraction between LT-α1/β2 ligand and cell surface LT-β receptors by50%. The ability to block tumor cell growth at least 20% identifies thissoluble LT-β receptor as a LT-β-R blocking agent according to thisinvention. As expected, the soluble TNF-R fusion protein (p55-TNF-R-Fc)completely blocked TNF-induced growth inhibition by binding to TNF andpreventing its interaction with surface receptor.

The soluble TNF-R fusion protein had no effect on LT ligand(LT-α1/β2)-mediated anti-proliferative effects. In contrast, the LT-β-Rfusion protein blocked LT ligand effects but not the effects of TNF orLT-α . Thus soluble human LT-β-R fusion proteins do not interfere withTNF-R activation by TNF and LT-α ligands.

To determine whether LT-β-R signalling is also cytotoxic to tumor cellsin mice, and whether soluble LT-β-R fusion proteins can blockLT-β-R-induced cytotoxicity, a similar experiment was performed usingmouse tumor cells. A soluble murine LT-β-R-Fc fusion protein(mLT-β-R-Fc; see Example 2) was tested for its ability to block thedeath of mouse WEHI 164 cells treated with LT ligand (Example 4).

FIG. 2 shows the effects of the soluble murine LT-β-R (mLT-β-R-Fc) on LTligand-induced LT-β-R signalling in mouse WEHI 164 cells. As this assayindicates, WEHI 164 cells are killed by treatment with soluble LT-α1/β2ligand. Addition of mLT-β-R-Fc blocks LT ligand-activated cell death.The control TNF receptor fusion protein (p55TNF-R-Fc) has little effecton blocking cell death.

These data show that a soluble LT-β-R fusion protein can effectivelycompete with surface LT-β-R molecules for LT ligand binding. The solublemLT-β-R-Fc fusion protein thus acts as a LT-β-R blocking agent in mice.

Source of Anti-Human LT-β-R Antibodies

In another embodiment of this invention, antibodies directed against thehuman LT-β receptor (anti-LT-β-R Abs) function as LT-β-R blockingagents. The anti-LT-β-R Abs of this invention can be polyclonal ormonoclonal (mAbs) and can be modified to optimize their ability to blockLT-β-R signalling, their in vivo bioavailability, stability, or otherdesired traits.

Polyclonal antibody sera directed against the human LT-β receptor areprepared using conventional techniques by injecting animals such asgoats, rabbits, rats, hamsters or mice subcutaneously with a human LT-βreceptor-Fc fusion protein (Example 1) in complete Freund's adjuvant,followed by booster intraperitoneal or subcutaneous injection inincomplete Freund's. Polyclonal antisera containing the desiredantibodies directed against the LT-β receptor are screened byconventional immunological procedures.

Mouse monoclonal antibodies (mAbs) directed against a human LT-βreceptor-Fc fusion protein are prepared as described in Example 5. Ahybridoma cell line (BD.A8.AB9) which produces the mouse anti-humanLT-β-R mAb BDA8 was deposited on Jan. 12, 1995 with the American TypeCulture Collection (ATCC) (10801 University Boulevard, Manassas, Va.20110-2209) according to the provisions of the Budapest Treaty, and wasassigned the ATCC accession number HB11798. All restrictions on theavailability to the public of the above ATCC deposits will beirrevocably removed upon the granting of a patent on this application.

Various forms of anti-LT-β-R antibodies can also be made using standardrecombinant DNA techniques (Winter and Milstein, Nature, 349, pp. 293-99(1991)). For example, "chimeric" antibodies can be constructed in whichthe antigen binding domain from an animal antibody is linked to a humanconstant domain (e.g. Cabilly et al., U.S. Pat. No. 4,816,567; Morrisonet al., Proc. Natl. Acad. Sci. U.S.A., 81, pp. 6851-55 (1984)). Chimericantibodies reduce the observed immunogenic responses elicited by animalantibodies when used in human clinical treatments.

In addition, recombinant "humanized antibodies" which recognize theLT-β-R can be synthesized. Humanized antibodies are chimeras comprisingmostly human IgG sequences into which the regions responsible forspecific antigen-binding have been inserted (e.g. WO 94/04679). Animalsare immunized with the desired antigen, the corresponding antibodies areisolated, and the portion of the variable region sequences responsiblefor specific antigen binding are removed. The animal-derived antigenbinding regions are then cloned into the appropriate position of humanantibody genes in which the antigen binding regions have been deleted.Humanized antibodies minimize the use of heterologous (inter-species)sequences in human antibodies, and are less likely to elicit immuneresponses in the treated subject.

Construction of different classes of recombinant anti-LT-β-R antibodiescan also be accomplished by making chimeric or humanized antibodiescomprising the anti-LT-β-R variable domains and human constant domains(CH1, CH2, CH3) isolated from different classes of immunoglobulins. Forexample, anti-LT-β-R IgM antibodies with increased antigen binding sitevalencies can be recombinantly produced by cloning the antigen bindingsite into vectors carrying the human μ chain constant regions(Arulanandam et al., J. Exp. Med., 177, pp. 1439-50 (1993); Lane et al.,Eur. J. Immunol., 22, pp. 2573-78 (1993); Traunecker et al., Nature,339, pp. 68-70 (1989)).

In addition, standard recombinant DNA techniques can be used to alterthe binding affinities of recombinant antibodies with their antigens byaltering amino acid residues in the vicinity of the antigen bindingsites. The antigen binding affinity of a humanized antibody can beincreased by mutagenesis based on molecular modelling (Queen et al.,Proc. Natl. Acad. Sci. U.S.A., 86, pp. 10029-33 (1989); WO 94/04679).

It may be desirable to increase or to decrease the affinity ofanti-LT-β-R Abs for the LT-β-R depending on the targeted tissue type orthe particular treatment schedule envisioned. For example, it may beadvantageous to treat a patient with constant levels of anti-LT-β-R Abswith reduced ability to signal through the LT-β pathway forsemi-prophylactic treatments. Likewise, inhibitory anti-LT-β-R Abs withincreased affinity for the LT-β-R may be advantageous for short-termtreatments.

Anti-LT-β-R Antibodies As LT-β-R Blocking Agents

Anti-LT-β-R antibodies that act as LT-β-R blocking agents may beselected by testing their ability to inhibit LT-β-R-induced cytotoxicityin tumor cells (Example 5).

In a preferred embodiment of this invention, compositions and methodscomprise the mouse anti-human LT-β-R mAb BDA8. FIG. 3 shows that mAbBDA8 acts as a LT-β-R blocking agent as defined by this invention. WiDrtumor cells stop growing in the presence of IFN-γ and soluble LT-α1/β2ligand. Control antibodies (IgG1 ) have no effect on this growthinhibition. In contrast, the anti-LT-β-R mAb BDA8 blocks the ability ofsoluble LT-α1/β2 ligand to inhibit WiDr cell growth. Thus an antibodydirected against human LT-β-R can function as a LT-β-R blocking agent asdefined by the present invention.

By testing other antibodies directed against the human LT-β receptor, itis expected that additional anti-LT-β-R antibodies that function asLT-β-R blocking agents in humans can be identified using routineexperimentation and the assays described herein.

Source of Anti-Surface LT Ligand Antibodies

Another preferred embodiment of this invention involves compositions andmethods which comprise antibodies directed against LT ligand thatfunction as LT-β-R blocking agents. As described above for theanti-LT-β-R Abs, anti-LT ligand antibodies that function as LT-β-Rblocking agents can be polyclonal or monoclonal, and can be modifiedaccording to routine procedures to modulate their antigen bindingproperties and their immunogenicity.

The anti-LT antibodies of this invention can be raised against eitherone of the two LT subunits individually, including soluble, mutant,altered and chimeric forms of the LT subunit. If LT subunits are used asthe antigen, preferably they are LT-β subunits. If LT-α subunits areused, it is preferred that the resulting anti-LT-α antibodies bind tosurface LT ligand and do not cross-react with secreted LT-α or modulateTNF-R activity (according to the assays described in Example 3).

Alternatively, antibodies directed against a homomeric (LT-β) or aheteromeric (LT-α/62 ) complex comprising one or more LT subunits can beraised and screened for activity as LT-β-R blocking agents. Preferably,LT-α1/β2 complexes are used as the antigen. As discussed above, it ispreferred that the resulting anti-LT-α1/β2 antibodies bind to surface LTligand without binding to secreted LT-α and without affecting TNF-Ractivity.

The production of polyclonal anti-human LT-α antibodies is described inapplicants' co-pending application (WO 94/13808). Monoclonal anti-LT-αand anti-LT-β antibodies have also been described (Browning et al., J.Immunol., 154, pp. 33-46 (1995)).

Mouse anti-human LT-β mAbs were prepared as described in Example 6. Ahybridoma cell line (B9.C9.1) which produces the mouse anti-human LT-β-RmAb B9 was deposited on Jul. 21, 1995 with the American Type CultureCollection (ATCC) (10801 University Boulevard, Manassas, Va. 20110-2209)according to the provisions of the Budapest Treaty, and was assigned theATCC accession number 11962.

Monoclonal hamster anti-mouse LT-α/62 antibodies were prepared asdescribed in Example 7. A hybridoma cell line (BB.F6.1) which producesthe hamster anti-mouse LT-α/62 mAb BB.F6 was deposited on Jul. 21, 1995with the American Type Culture Collection (ATCC) (10801 UniversityBoulevard, Manassas, Va. 20110-2209) according to the provisions of theBudapest Treaty, and was assigned the ATCC accession number MB11963.

All restrictions on the availability to the public of the above ATCCdeposits will be irrevocably removed upon the granting of a patent onthis application.

Anti-LT Ligand Antibodies As LT-β-R Blocking Agents

A fluorescence-activated cell sorting (FACS) assay was developed toscreen for antibodies directed against LT subunits and LT complexes thatcan act as LT-β-R blocking agents (Examples 6 and 7). In this assay,soluble human LT-β-R-Fc fusion protein is added to PMA-activated II-23cells--which express surface LT complexes (Browning et al., J. Immunol.,154, pp. 33-46 (1995))--in the presence of increasing amounts of thetest antibody. An antibody that can inhibit LT-β receptor-ligandinteraction by at least 20% is selected as a LT-β-R blocking agent.

The results of this assay performed to test the mouse anti-human LT-βmAb B9 are shown in FIG. 4. FIG. 4 shows that anti-LT-β mAb B9 canselectively block the binding of soluble LT-β-R-Fc fusion proteins tosurface LT ligands induced on activated cells. These results confirmthat antibodies directed against a LT ligand subunit will function as anLT-β-R blocking agent.

The FACS assay described above was also used to test mAbs raised inhamster against a soluble mouse LT-α/62 complex (Example 7). The resultsof this assay performed to test the hamster anti-mouse LT-α/62 mAb BB.F6are shown in Table 2 (Example 7). Table 2 shows that anti-LT-α/62 mAbBB.F6 can effectively block the binding of soluble mLT-β-R-Fc fusionproteins (Example 2) to surface LT ligands expressed on a murine T cellhybridoma and is thus a LT-3-R blocking agent according to thisinvention.

Using a LT-α/β complex rather than a LT subunit as an antigen toimmunize an animal may lead to more efficient immunization, or mayresult in antibodies having higher affinities for surface LT ligand. Itis conceivable that by immunizing with the LT-α/62 complex, antibodieswhich recognize amino acid residues on both the LT-α and the LT-βsubunits (e.g., residues that form an LT-α/62 cleft) can be isolated.

By testing antibodies directed against human LT-α/62 heteromericcomplexes, it is expected that additional anti-LT antibodies thatfunction as LT-β-R blocking agents in humans can be identified usingroutine experimentation and the assays described herein.

LT-β-R Blocking Agents Inhibit Th1 Cell-Mediated ContactHypersensitivity in mouse

The LT-β-R blocking agents of this invention can inhibit Th1cell-mediated immune responses. One such Th1-mediated response isdelayed type hypersensitivity (DTH; Cher and Mosmann, J. Immunol., 138,pp. 3688-94 (1987); see also I. Roitt et al., Immunology, pp.22.1-22.12, Mosby-Year Book Europe Ltd., 3d ed. (1993) for a generaldiscussion). DTH is evoked when antigen-sensitized Th1 cells secretecytokines following a secondary contact with the same antigen. The Th1cytokines attract and activate macrophages that release additionaleffector molecules which trigger inflammatory reactions.

DTH reactions are classified into three different types: contacthypersensitivity, tuberculin-type hypersensitivity and granulomatousreactions. The three types of hypersensitivity (HS) may be distinguishedby the speed and nature of the response to foreign antigen when it isapplied directly to or injected beneath the skin of a sensitizedsubject. The DTH reaction is monitored by measuring the rate and degreeto which the skin thickens.

Tuberculin-type HS reactions are skin reactions which occur at theinjection site of a foreign antigen from a microorganism to which thesubject has been previously exposed (e.g. mycobacterium tuberculosis orM. leprae). This skin reaction, which is maximal between 48 and 72hours, is frequently used as the basis for diagnostic sensitivity teststo previously-encountered microorganisms (e.g. the tuberculin skintest). As a tuberculin-type lesion develops, it can become agranulomatous reaction if the antigen persists in the tissue.

Granulomatous reactions are clinically the most serious DTH reactionsbecause they can lead to many of the pathological effects associatedwith Th1 cell-mediated diseases. Granulomatous reactions occur whenantigens or immune complexes fail to clear from macrophages and continueto stimulate Th1 cytokine secretion. Chronic inflammation andaggregation of activated macrophages at the site of the stimuluscharacterize granulomatous reactions.

A core of epithelial cells and macrophages, which can also be surroundedby lymphocytes and fibrotic depositions, form a hardened structurecalled a granuloma. Sometimes there is extensive cell death in the coreof the granuloma (e.g. in tuberculosis-affected lung tissue). Hardeningin the target tissue of a granulomatous reaction occurs in about 4weeks.

Agents which affect the frequency of granuloma formation can beidentified using schistosome-infected mice (Amiri et al., Nature, 356,pp. 604-607 (1992)). Schistosome worms (blood flukes) can cause aparasitic disease leading to granuloma formation around the schistosomeeggs deposited in portal venules of the infected liver. Agents thatinhibit this Th1 cell-mediated DTH response may decrease the size of thegranuloma, or the frequency or rate of granuloma formation inschistosome-infected mouse livers. Cellular reaction to the schistosomeeggs can be assessed by quantitating the number and size of granulomasformed in mice treated with increasing concentrations of a putativeLT-β-R blocking agent over time.

Contact hypersensitivity (CHS) is a class of DTH in which skin is thetarget organ. In CHS, an inflammatory response is caused by locallyapplying a reactive hapten onto the skin. Allergens generally compriseat least one hapten molecule, which is usually too small to be antigenicon its own. The hapten penetrates the epidermis and reacts with a normalprotein under the skin to produce a novel antigenic complex.

Re-exposure of a sensitized subject to the hapten triggers the DTHresponse. The hapten-carrier protein conjugate, in combination withantigen presenting cells, activates effector mechanisms that trigger therelease of cytokines (including IL-2, IL-3, IFN-γ and GM-CSF). Thecascade of released cytokines causes CD4+ T cells to proliferate, theexpression patterns of various cell surface adhesion molecules tochange, and the attraction of T cells and macrophages to the skin at thesite of inflammation.

The cytokine cascade and resulting vasodilation, cellular infiltrationand edema of the dermis and epidermis leads to swelling and inflammationof the target tissue, which accounts for the measurable skin thickeningin response to DTH reactions.

The degree to which a particular hapten can sensitize an individualdepends on a variety of factors. These factors include how well thehapten can penetrate the skin and react with a host carrier protein toform a conjugate. One hapten that sensitizes nearly all individuals is2,4-dinitro-fluorobenzene (DNFB).

The skin CHS response to a hapten such as DNFB is a classic animal modelfor cell-mediated immunity. Localization of this CHS response to the earof a sensitized mouse allows easy, accurate and reproduciblequantitation of this cell-mediated immune response in vivo by measuringear thickness. The details of the murine CHS reaction and thehistopathology of the DNFB-induced inflammatory response have beenreported (Chisholm et al., Eur. J. Immunol., 23, pp. 682-688 (1993)).

The ability of DNFB to induce a contact hypersensitivity response inmost individuals can be used to identify agents that reduce or eliminatethe inflammatory responses associated with Th1 cell-mediated DTHreactions. A soluble murine LT-β-R-Fc fusion protein effectivelyinhibits DNFB-induced contact hypersensitivity responses in mice(Example 8).

Mice were initially sensitized by applying DNFB onto the bottom of eachhind foot on two consecutive days. Five days after the initialsensitization, a sub-irritant dose of DNFB in carrier solution wasapplied to the surfaces of the left ear. Carrier solution alone wasapplied to the right ear as a control.

Increasing concentrations of the LT-β-R blocking agent mLT-β-R-Fc(Example 2) were then injected intravenously into the mice (Example 8).Injections of PBS buffer alone, or of a human IgG fusion protein(LFA3-Fc) served as negative controls, and injection of ananti-VLA4-specific mAb (PS/2 mAb) known to inhibit CHS served as apositive control.

Twenty-four hours after challenge, the thickness of each ear(DNFB-challenged and -unchallenged) was measured. Inhibition of the earswelling response by the LT-β-R blocking agent was judged by comparisonof treated groups with their negative control group.

FIG. 5 shows that mLT-β-R-Fc causes a significant reduction in the earswelling response of DNFB-treated mice compared to uninhibitedDNFB-treated control animals (PBS and LFA3-Fc). Soluble LT-β-R can blockthis CHS reaction as effectively as the inhibitor anti-VLA4-specific mAb(PS/2 mAb), which acts by blocking the influx of T cells into thechallenge site (Chisholm et al., Eur. J. Immunol., 23, pp. 682-88(1993)).

These data show that a soluble LT-β-R fusion protein which acts as aLT-β-R blocking agent in vitro can also effectively inhibit a Th1cell-mediated immune response when administered to an animal. The LT-β-Rblocking agents of this invention identified in vitro can be testedusing this ear swelling assay, or other DTH assays such as thosedescribed above, to select additional LT-β-R blocking agents that willbe useful for reducing the severity of Th1 cell-associated immuneresponses in vivo.

LT-β-R Blocking Agents Do Not Inhibit a Th2 Cell-Mediated (Humoral)Immunological Response

As shown above, the LT-β-R blocking agents of this invention can inhibita Th1 cell-mediated effector mechanism such as contact delayed typehypersensitivity (FIG. 5). This Th1 cell-mediated response is inhibitedwithout significantly affecting Th2 cell-dependent responses. Thedifferential effect of LT-β-R blocking agents on Th1 cell-mediatedimmune responses was shown by monitoring a Th2-cell dependent immuneresponse--such as a primary antibody response and isotype switching--inthe presence of an LT-β-R blocking agent.

Mice were injected five times over the course of a ten day period witheither soluble LT-β-R fusion protein (mLT-β-R-Fc; Example 2) or controlIgG fusion protein (LFA3-Fc), or were left untreated. After the secondinjection, all mice were injected in the base of the tail with 100 μl ofcomplete Freund's adjuvant containing 100 μg of ovalbumin. After 11days, primary serum anti-ovalbumin-specific antibody titers wereanalyzed using an ELISA specific for IgG1 , IgG2a and IgM isotypes.

FIG. 6 shows the effect of the mouse LT-β-R blocking agent mLT-β-R-Fc onserum anti-ovalbumin antibody production in mice immunized withovalbumin (Example 9). Administering the LT-β-R blocking agent does notsignificantly affect primary antibody titers following ovalbuminimmunization. By comparison, interfering with CD40 ligand-induced CD40receptor signalling completely blocks the antigen-specific IgG responsein mice (Renshaw et al., J. Exp. Med., 180, pp. 1889-1900 (1994)). CD40is another ligand/receptor pair in the TNF family.

Total immunoglobulin maturation is maturation is clearly Th2cell-dependent. However, there is also evidence that the Th1 cytokineIFN-γ participates but is not absolutely required for the switch to theIgG2a subclass (Huang et al., Science, 259, pp. 1742-45 (1993)). TheLT-β-R blocking agent mLT-β-R-Fc did not inhibit the IgG2a switch inthese experiments. It is possible that the LT-β-R blocking agents ofthis invention do not block this humoral aspect of a Th1 cell-mediatedresponse. In addition, the proliferatory responses of lymphocytes fromthe mLT-β-R-Fc-treated mice were not decreased (Example 10; FIG. 7).

These experiments indicate that a therapy based on administering theLT-β-R blocking agents of this invention will not adversely affect Th2dependent antibody production functions of an immune response. Thenormal pattern of antibody response illustrated in FIG. 6 also indicatesthat an intensive treatment with soluble mLT-β-R-Fc was not toxic to themice, further indicating the useful therapeutic nature of thecompositions and methods set forth in this invention.

T Helper Cell-Mediated Diseases

Many organ-specific autoimmune conditions appear to involve pathologicalTh1 response. These data have been reviewed (Modlin and Nutman, CurrentOpinion in Immunol., 5, pp. 511-17 (1993); Romagnani et al., Ann. Rev.Immunol., 12, pp. 227-57 (1994)). These organ-specific autoimmuneconditions include: multiple sclerosis, insulin-dependent diabetes,sympathetic ophthalmia, uveitis and psoriasis.

Insulin-dependent diabetes mellitus is an autoimmune disease in whichthe insulin-producing beta pancreatic cells are destroyed by leukocytesinfiltrating into the islets of Langerhans. Diabetes can be rapidlyinduced in neonatal nonobese diabetic (NOD) mice by transferringactivated prediabetic splenocytes. Recently, Th1- or Th2-like cells,otherwise genetically similar, were transferred into neonatal NOD mice.Only the Th1 cells rapidly induced diabetes--and in almost allrecipients (Katz et al., Science, 268, pp. 1185-88 (1995)). Thisindicates that the LT-β-R blocking agents of this invention--which caninhibit the effects of a Th1 cell-mediated immune response in vivo--willbe useful for treating or preventing insulin-dependent diabetes.

Several systemic autoimmune diseases, including various arthritides, areTh1 cell-associated. Rheumatoid arthritis and Sjorgren's syndrome bothappear to involve Th0 and Th1 cells. In contrast, systemic lupuserythematosus (SLE) appears to have an aberrant Th0/Th2 dominatedresponse.

Some chronic inflammatory diseases also appear to have an aberrant Th1type response, including inflammatory bowel disease, sarcoidosis of thelung and allograft rejection. In general, the exact contribution ofauto-antibodies versus specific T cells has not been delineated in theseautoimmune diseases. Cellular responses may make major contributions topathogenicity in those systemic autoimmune diseases currently thought tobe primarily antibody driven, e.g. the various arthritides.

The normal immune response to some pathogenic infectious agents alsoelicits a Th1 response that can become excessive and present itself as amedical problem. Examples of granulomatous reactions (a class of DTHresponse described above) that lead to severe medical problems includeleprosy, granuloma formation in the lungs of tuberculosis patients,sarcoidosis and schistosomiasis (Roitt et al., Immunology, pp. 22.5-6(Mosby-Year Book Europe Ltd., 3d ed. 1993). Psoriasis is also likely tobe mediated by Th1 cells.

Cytolytic T cells, i.e. CTLs (CD8 positive T cells) may also subdivideinto Th1- and Th2-like populations. Therefore it is possible that muchof what is known regarding the Th groups will also apply to CD8+ cells,which are primarily involved in anti-viral and grafted tissue rejectionresponses.

Treatments Using LT-β-R Blocking Agents

The compositions of this invention will be administered at an effectivedose to treat the particular clinical condition addressed. Determinationof a preferred pharmaceutical formulation and a therapeuticallyefficient dose regiment for a given application is well within the skillof the art taking into consideration, for example, the condition andweight of the patient, the extent of desired treatment and the toleranceof the patient for the treatment. Doses of about 1 mg/kg of a solubleLT-β-R are expected to be suitable starting points for optimizingtreatment doses.

Determination of a therapeutically effective dose can also be assessedby performing in vitro experiments that measure the concentration of theLT-β-R blocking agent required to coat target cells (LT-β-R or LTligand-positive cells depending on the blocking agent) for 1 to 14 days.The receptor-ligand binding assays described herein can be used tomonitor the cell coating reaction. LT-β-R or LT ligand-positive cellscan be separated from activated lymphocyte populations using FACS. Basedon the results of these in vitro binding assays, a range of suitableLT-β-R blocking agent concentrations can be selected to test in animalsaccording to the assays described herein.

Administration of the soluble LT-β-R molecules, anti-LT ligand andanti-LT-β-R Abs of this invention, alone or in combination, includingisolated and purified forms of the antibodies or complexes, their saltsor pharmaceutically acceptable derivatives thereof, may be accomplishedusing any of the conventionally accepted modes of administration ofagents which exhibit immunosuppressive activity.

The pharmaceutical compositions used in these therapies may also be in avariety of forms. These include, for example, solid, semi-solid andliquid dosage forms such as tablets, pills, powders, liquid solutions orsuspensions, suppositories, and injectable and infusible solutions. Thepreferred form depends on the intended mode of administration andtherapeutic application. Modes of administration may include oral,parenteral, subcutaneous, intravenous, intralesional or topicaladministration.

The soluble LT-β-R molecules, anti-LT ligand and anti-LT-β-R Abs of thisinvention may, for example, be placed into sterile, isotonicformulations with or without cofactors which stimulate uptake orstability. The formulation is preferably liquid, or may be lyophilizedpowder. For example, the soluble LT-β-R molecules, anti-LT ligand andanti-LT-β-R Abs of this invention may be diluted with a formulationbuffer comprising 5.0 mg/ml citric acid monohydrate, 2.7 mg/ml trisodiumcitrate, 41 mg/ml mannitol, 1 mg/ml glycine and 1 mg/ml polysorbate 20.This solution can be lyophilized, stored under refrigeration andreconstituted prior to administration with sterile Water-For-Injection(USP).

The compositions also will preferably include conventionalpharmaceutically acceptable carriers well known in the art (see forexample Remington's Pharmaceutical Sciences, 16th Edition, 1980, MacPublishing Company). Such pharmaceutically acceptable carriers mayinclude other medicinal agents, carriers, genetic carriers, adjuvants,excipients, etc., such as human serum albumin or plasma preparations.The compositions are preferably in the form of a unit dose and willusually be administered one or more times a day.

The pharmaceutical compositions of this invention may also beadministered using microspheres, liposomes, other microparticulatedelivery systems or sustained release formulations placed in, near, orotherwise in communication with affected tissues or the bloodstream.Suitable examples of sustained release carriers include semipermeablepolymer matrices in the form of shaped articles such as suppositories ormicrocapsules. Implantable or microcapsular sustained release matricesinclude polylactides (U.S. Pat. No. 3,773,319; EP 58,481), copolymers ofL-glutamic acid and ethyl-L-glutamate (Sidman et al., Biopolymers, 22,pp. 547-56 (1985)); poly(2-hydroxyethyl-methacrylate) or ethylene vinylacetate (Langer et al., J. Biomed. Mater. Res., 15, pp. 167-277 (1981);Langer, Chem. Tech., 12, pp. 98-105 (1982)).

Liposomes containing soluble LT-β-R molecules, anti-LT ligand andanti-LT-β-R Abs of this invention, alone or in combination, can beprepared by well-known methods (See, e.g. DE 3,218,121; Epstein et al.,Proc. Natl. Acad. Sci. U.S.A., 82, pp. 3688-92 (1985); Hwang et al.,Proc. Natl. Acad. Sci. U.S.A., 77, pp. 4030-34 (1980); U.S. Pat. Nos.4,485,045 and 4,544,545). Ordinarily the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. % cholesterol. The proportion of cholesterolis selected to control the optimal rate of soluble LT-β-R molecule,anti-LT ligand and anti-LT-β-R Ab release.

The soluble LT-β-R molecules, anti-LT ligand and anti-LT-β-R Abs of thisinvention may also be attached to liposomes containing other LT-β-Rblocking agents, immunosuppressive agents or cytokines to modulate theLT-β-R blocking activity. Attachment of LT-β-R molecules, anti-LT ligandand anti-LT-β-R Abs to liposomes may be accomplished by any knowncross-linking agent such as heterobifunctional cross-linking agents thathave been widely used to couple toxins or chemotherapeutic agents toantibodies for targeted delivery. conjugation to liposomes can also beaccomplished using the carbohydrate-directed cross-linking reagent4-(4-maleimidophenyl) butyric acid hydrazide (MPBH) (Duzgunes et al., J.Cell. Biochem. Abst. Suppl. 16E 77 (1992)).

Advantages of therapeutic compositions comprising LT-β-R blocking agents

The LT-β-R blocking agents of this invention are capable of selectivelyinhibiting Th1 and not Th2 cell-dependent immune effector mechanisms.LT-β-R blocking agents will be useful in treating conditions that areexacerbated by the activities of Th1-type cytokines (e.g., IL-2 andIFN-γ). Because Th1 cytokines can inhibit Th2 cell-dependent responses,LT-β-R blocking agents may also indirectly stimulate certain Th2cell-dependent responses that are normally inhibited by Th1-inducedcytokine cascades.

The ability to selectively suppress Th1 (or indirectly stimulate Th2)cell responses will be useful for treating abnormalities in diversecell-mediated immune responses including various autoimmune and chronicinflammatory conditions, antigen tolerance, and cellular rejection oftissue grafts and organ transplants.

As discussed above, treatment of Th1 cell-based immunological conditionsgenerally employs immunomodulatory and immunosuppressive agents whichhave pleiotropic effects on a wide variety of cell types andimmunological responses. These non-specific immunosuppressive agents aregenerally required in high and often cytotoxic doses that cause adverseside effects.

The ability to shift the character of an immunological response issupported in the recent study of mouse diabetes discussed above (Katz etal., Science, 268, pp. 1185-88 (1995)), and in an allogeneic transplantmodel (Sayegh et al., J. Exp. Med., 181, pp. 1869-74 (1995)). In thelatter study, a fusion protein that blocks the CD28-B7 T cellcostimulatory pathway was shown to induce renal graft tolerance. Thetolerance correlated with a decrease in Th1 cytokines and an increase inTh2 cytokines in vivo. These data indicate that the LT-β-R blockingagents of this invention will be useful in suppressing cellularrejection of tissue grafts and organ transplants by inhibiting Th1cell-mediated cytokine release.

The LT-β-R blocking agents of the compositions and methods of thisinvention can be modified to obtain a desirable level of LT-β-Rsignalling depending on the condition, disorder or disease beingtreated. It is envisioned that the absolute level of LT-β-R signallingcan be fine-tuned by manipulating the concentration and the affinitiesof the LT-β-R blocking agents for their respective molecular targets.

For example, in one embodiment of this invention, compositionscomprising soluble LT-β-R molecules are administered to a subject. Thesoluble LT-β receptor can effectively compete with cell surface LT-βreceptors for binding surface LT ligands. The ability to compete withsurface LT ligands depends on the relative concentrations of the solubleand the cell surface LT-β-R molecules, and on their relative affinitiesfor ligand binding.

Soluble LT-β-R molecules harboring mutations that increase or decreasethe binding affinity of that mutant soluble LT-β-R with surface LTligand can be made using standard recombinant DNA techniques well knownto those of skill in the art. Large numbers of molecules withsite-directed or random mutations can be tested for their ability to actas LT-β-R blocking agents using routine experimentation and thetechniques described herein.

Similarly, in another embodiment of this invention, antibodies directedagainst either the LT-β receptor or one or more of the LT ligandsubunits function as LT-β-R blocking agents. The ability for theseantibodies to block LT-β receptor signalling can be modified bymutation, chemical modification or by other methods that can vary theeffective concentration or activity of the antibody delivered to thesubject.

The ability to diminish LT-β-R signalling without completely inhibitingit may be important for establishing or maintaining reduced levels ofLT-β-R signalling that support normal immune function while inhibitingTh1-cell mediated responses which are exaggerated or abnormal.

Disruption of the LT-α gene in a mouse leads to aberrant peripherallymphoid organ development (De Togni et al., Science, 264, pp. 703-7(1994)). Such mice lacked lymph nodes and their spleens lacked theusually clear demarcation between T and B cell-rich regions in thefollicles. We believe that this phenotype is associated with loss ofsurface LT-induced LT-β-R signalling because similar phenotypes have notbeen observed by modulating TNF-R activity. The ability to selectivelyor to partially block the LT-β-R pathway may thus be useful in treatingabnormal lymphoid organ development associated with mis- orover-expression of signalling by the LT-β-R pathway.

Some Th1-associated reactions are critical components of a number ofcell-mediated immune responses (Romagnani, S., Ann. Rev. Immunol., 12,pp. 227-57 (1994)), and absolute inhibition of Th1 cell activity may notbe desirable in certain circumstances. For example, a mouse caneffectively resist a parasitic infection when a good Th1 response can bemounted. Infectious agents such as Listeria and Toxoplasma also elicitstrong Th1-type responses. In humans, mycobacterium tuberculosisresponses appear to be Th1-based. Leishmaniasis pathogenicity correlateswith responses similar to the Th1 responses characterized in mouse (Reedand Scott, Current Opinion in Immunol., 5, pp. 524-31 (1993)).

The ability to influence the level of Th1 inhibition by blocking LT-β-Rsignalling may be important in maximizing the beneficial results whichcan be achieved by treatments with the LT-β-R blocking agents of thisinvention.

The following are examples which illustrate the soluble LT-β receptors,anti-LT ligand and anti-LT-β-R antibodies of this invention and themethods used to characterize them. These examples should not beconstrued as limiting: the examples are included for purposes ofillustration and the present invention is limited only by the claims.

EXAMPLE 1 Preparation of Soluble Human LT-β Receptors as ImmunoglobulinFc Fusion Proteins

The sequence of a human cDNA clone isolated from a library of human 12ptranscribed sequences derived from a somatic cell hybrid (Baens et al.,Genomics, 16, pp. 214-18 (1993)), was entered into GenBank and was lateridentified as the sequence which encodes human LT-β-R. The sequence ofthis full-length human LT-β-R cDNA clone has been available since 1992as GenBank entry L04270.

The extracellular domain of LT-β-R up to the transmembrane region(FIG. 1) was amplified by PCR from a cDNA clone using primers thatincorporated NotI and SalI restriction enzyme sites on the 5' and 3'ends, respectively (Browning et al., J. Immunol., 154, pp. 33-46(1995)). The amplified product was cut with NotI and SalI, purified andligated into a NotI-linearized vector pMDR901 along with a SalI-NotIfragment encoding the Fc region of human IgG1 . The resultant vectorcontained the dihydrofolate reductase gene and the LT-β-R-Fc fusionprotein driven by separate promoters.

The vector was electroporated into CHO dhfr⁻ cells andmethotrexate-resistant clones were isolated as per standard procedures.The LT-β-R-Fc was secreted into the medium and an ELISA assay was usedto select for cell lines producing the highest level of the receptorfusion protein. A high-producing cell line was grown to large numbersand the conditioned medium collected. The pure LT-β receptor fusionprotein was isolated by Protein A Sepharose Fast Flow affinitychromatography (Pharmacia).

EXAMPLE 2 Preparation of Soluble Murine LT-β Receptors as ImmunoglobulinFc Fusion Proteins

A complete cDNA clone of the mLT-β-R was prepared by ligating a 5'NotI/ApaLI and 3' ApaLI/NotI fragments from two partial cDNA isolatesinto the NotI site of pCDNA3 (InVitrogen, San Diego, Calif.). Thesequence of this cDNA clone is accessible as GenBank entry U29173. Nocoding sequence differences were noted when compared with anothersequence entry for mLT-β-R found in GenBank entry L38423.

A soluble mLT-β-R (hIgG1 ) fusion protein was prepared by PCRamplification of the full length mLT-β-R cDNA clone as a template andthe primers 5'AACTGCAGCGGCCGCCATGCGCCTGCCC 3' and5'GACTTTGTCGACCATTGCTCCTGGCTCTGGGGG 3'. The amplified product waspurified and cut with NotI and SalI and ligated with a SalI/NotI humanIgG1 Fc fragment into NotI-linearized and phosphatase-treated SAB132 toform JLB 122. For stable expression, the NotI cassette containing themLT-β-R-Fc fragment was transferred into the NotI site of pMDR901forming PSH001 and the vector was transfected into CHO cells asdescribed (Browning et al., J. Immunol., 154, pp. 33-46 (1995)). Cellclones secreting mLT-β-R-Fc were identified by ELISA analysis. Thepurified receptor fusion protein was isolated from CHO cell supernatantsby Protein A Sepharose Fast Flow chromatography (Pharmacia).

EXAMPLE 3 Use of Soluble Human LT-β-R-Fc to Block LT-β Receptor-LigandInteractions

Soluble hLT-β-R-Fc was tested for its ability to block LT ligand bindingto the LT-β receptor in the tumor cell cytotoxicity assay describedabove. In this assay, a soluble form of the LT ligand (hLT-α1/β2), whichactivates LT-β-R signalling, is used to kill human tumor cells.Inhibitors of LT-β-R signalling can reduce LT-β-R-induced tumor cellcytotoxicity.

Soluble LT-α1/β2 ligands comprise truncated or modified LT-β subunitslacking a functional transmembrane domain. Soluble LT-α1/β2 ligands bindto and stimulate LT-β-R signalling as well as surface forms of LT ligand(Browning et al., J. Immunol., 154, pp. 33-46 (1995)).

Serial dilutions of hLT-α1/β2, hTNF or hLT-α were prepared in 0.05 ml in96 well plates and 5000 trypsinized HT29 cells (ATCC) added in 0.05 mlmedia containing 80 U/ml (antiviral units) of hu-IFN-γ. After 4 days,mitochondrial reduction of the dye MTT was measured as follows: 10 μl ofMTT was added and after 3 hours, the reduced dye dissolved with 0.09 mlof isopropanol with 10mM HCl, and the O.D. measured at 550 nm. Solublereceptor forms or pure human IgG were added in 10 μl prior to theaddition of the cells to give a final concentration of 5 μg/ml.

Table 1 compares the ability of hLT-β-R-Fc and p55-TNF-R-Fc chimeras(with human IgG as a control) to block the inhibitory effects of varioussoluble TNF and LT ligands on HT29 tumor cell growth.

                  TABLE I    ______________________________________    Ability of LT-β-R and p55-TNF-R Immunoglobulin Fusion    Proteins to Block the Inhibitory Effects of Various TNF    and LT Ligands on HT29 Growth    Concentration of Cytotoxic Agent    (ng/ml) Resulting in 50% Growth Inhibition            In the Presence of.sup.a    Cytotoxic Agent              hu-IgG control                          p55-TNF-R-Fc                                      LT-β-R-Fc    ______________________________________    TNF       0.08          >10.sup.b 0.08    LT-α              3           >1000       3    LT-α1/β2              5             5         >200    ______________________________________     .sup.a Each cytotoxic agent was premixed with the Ig fusion proteins for     10 minutes prior to addition to the cells. The final concentration of     fusion protein was 5 μg/ml.     .sup.b Higher concentrations were not tested.

The data in Table 1 indicate that the soluble human LT-β-R fusionprotein (hLT-β-R-Fc) can effectively block the interaction between LTligand (LT-α1/β2) and cell surface LT-β receptors and is thus a LT-β-Rblocking agent according to this invention.

As expected, the soluble TNF-R fusion protein (p55-TNF-R-Fc) completelyblocked TNF-induced growth inhibition by binding to TNF and preventingits interaction with surface TNF receptors. This soluble TNF receptorhad no effect on LT ligand-mediated anti-proliferative effects. Incontrast, the LT-β-R-Fc blocked LT ligand-induced cytotoxic effects butnot those of TNF or LT-α . Thus soluble human LT-β-R fusion proteins donot interfere with TNF-R activation by TNF and LT-α ligands.

EXAMPLE 4 Use of Soluble Murine LT-β-R-Fc to Block Mouse LT-βReceptor-Ligand Interactions

A soluble murine LT-β receptor coupled to a human IgG1 Fc domain(mLT-β-R-Fc; see Example 2) was tested for its ability to block LT-βreceptor-ligand interaction in mouse using a cytotoxicity assay on mousecells (FIG. 2). The cytotoxicity assay was performed on WEHI 164 cellsusing essentially the same procedure as was used in the HT29 cell assaydescribed in Example 3 (see also Browning and Ribolini, J. Immunol.,143, pp. 1859-67 (1989)).

FIG. 2 shows the effects of mLT-β-R-Fc on ligand-induced LT-β-Rsignalling in mouse WEHI 164 cells. As this assay indicates, WEHI 164cells are killed by treating them with LT-α/62 ligand at concentrationsranging from about 1 to 100 ng/ml. Soluble mLT-β-R-Fc (10 μ/ml) blocksthe LT ligand-activated cell death. Adding a soluble mouse p55-TNF-R-Fcfusion protein or IgG control antibodies (each at 10μ/ml) had little orno effect on blocking cell death.

These data show that the mLT-β-R-Fc fusion protein can effectivelycompete with surface LT-β-R molecules for LT ligand binding. These dataalso show that LT-α/62 -induced cytotoxicity is LT-β-R-mediated and canbe inhibited by soluble mLT-β-R-Fc, which acts as a LT-β-R blockingagent according to the present invention.

EXAMPLE 5 Use of Anti-Human LT-β-R Antibodies to Block LT-βReceptor-Ligand Interactions

Mouse monoclonal antibodies (mAbs) directed against the human LT-βreceptor were prepared by intraperitoneal immunization of RBF micerepetitively with a CHO cell-derived hLT-β-R-Fc fusion protein attachedto Protein A Sepharose beads in the absence of adjuvant. Animals werefinally boosted with soluble hLT-β-R-Fc, both i.p. and i.v., spleencells were fused via classical protocols and hybridoma supernatants werescreened by ELISA (Ling et al., J. Interferon and Cytokine Res., 15, pp.53-59 (1995)). Hybridoma supernatants were screened further for theirability to block binding of activated II-23 hybridoma cells--whichexpress surface LT-α1/β2--to LT-β-R-Fc coated plates in a cell panningassay. Pure mAbs were prepared by Protein A Sepharose purification(Pharmacia) of IgG from culture supernatants.

To determine whether an anti-LT-β receptor mAb could block LT-β-Rsignalling initiated by the binding of soluble LT, a tumor cellcytotoxicity assay was performed using WiDr human carcinoma cells. Inthe cytotoxicity assays, serial dilutions of LT-α1/β2 were prepared in0.05 ml in 96 well plates and 10 μl of a 100 μg/ml solution containingeither control mouse IgG1 mAb or the anti-LT-β receptor mAb was added.5000 trypsinized WiDr cells (ATCC) were then added to each well in 0.05ml of media containing 50 U/ml (antiviral units) of hu-IFN-γ. After 4days, mitochondrial reduction of the dye MTT was measured as follows: 10μl of MTT was added and after 3 hours, the reduced dye dissolved with0.09 ml of isopropanol with 10 mM HCl, and the O.D. measured at 550 nm.The amount of purple color is proportional to the amount of cell growth.

FIG. 3 shows that the anti-LT-β-R mAb BDA8 acts as a LT-β-R blockingagent according to this invention. Human WiDr carcinoma cells stopgrowing in the presence of IFN-γand soluble LT-α1/β2 ligand (from about0.05 to 50 ng/ml). An IgG1 control antibody (10 μg/ml) has no effect onthis growth inhibition. In contrast, the anti-LT-β-R mAb BDA8 (10 μg/ml)restores the ability of WiDr cells to grow in the presence of solubleLT-α1/β2 ligand.

EXAMPLE 6 Use of Anti-Human LT-β Antibodies to Block Receptor-LigandInteractions

Anti-human LT-β mAbs were prepared by immunizing RBF mice with washedprotein A Sepharose-9E10-rLT-β beads containing about 1-2 μg of humanrecombinant LT-β in CFA, and followed with one boost of the samematerial in IFA. Eight weeks after the last boost, mice were given i.v.30 μg of purified soluble rLT-β (acid eluted off the 9E10 resin) and 20μg of the same soluble material 2 days later. One day after the secondi.v. boost, the spleen cells were fused using classical protocols tocreate mAbs. Hybridoma supernatants were screened directly by ELISA orby FACS staining of PMA-activated II-23 cells. Pure mAbs were preparedby Protein A Sepharose Fast Flow purification of IgG from culturesupernatants (Pharmacia).

A FACS assay was used to select antibodies directed against LT-β thatcan effectively block the binding of soluble LT-α/62 ligand to LT-βreceptors on the surface of a cell--thus mimicking the interactionbetween two cells in vivo. In this assay, soluble human LT-β-R-Fc (2μg/ml) was allowed to bind to surface LT ligand on PMA-activated II-23cells (Browning et al., J. Immunol., 154, pp. 33-46 (1995)) in thepresence of increasing concentrations of the test anti-LT-β mAb (0.02-20μg/ml). The cells were washed and the bound LT-β-R-Fc was detected byreaction with phycoerythrin-labelled donkey anti-human IgG. The amountof bound fluorescent label was determined by FACS analysis and the meanfluorescence intensity was plotted.

FIG. 4 shows the results of a FACS assay which measured the ability ofthe anti-LT-β mAb B9 to block LT-β receptor-ligand interaction asdescribed above. This experiment shows that the anti-LT-β mAb B9 (0.02-5μg/ml) can specifically and effectively compete for cell surface LTligand binding with soluble LT-β-R fusion protein (2 μg/ml) and thusqualifies as an LT-β-R blocking agent according to this invention.

EXAMPLE 7 Use of Anti-Mouse LT-α/62 Antibodies to Block Receptor-LigandInteractions

Soluble mouse LT-α/β complexes were prepared as described above for thehuman soluble LT-α/62 complexes. The soluble mouse LT-β subunit was madebased on sequence information previously described (Lawton et al., J.Immunol., 154, pp. 239-46 (1995)).

Soluble murine LT-α/0 complexes were expressed using thebaculovirus/insect cell expression system and the LT-α/β complexes wereisolated by affinity chromatography using human p55 TNF-R and LT-β-Rcolumns essentially as described above for the expression andpurification of human LT-α/β complexes.

Armenian hamsters were immunized with purified soluble murine LT-α/62complex essentially as described in Example 6. Hamster spleen cells werefused to the mouse P3X hybridoma cell line as described (Sanchez-Madridet al., Methods of Enzymology, 121, pp. 239-44 (1986)). Hybridomas weregrouped as anti-mLT-β or anti-mLT-α on the basis of their bindingcharacteristics to either the LT-α/β complex or to LT-α alone,respectively. Hybridoma cells were expanded and the antibodies purifiedfrom the culture supernatant using Protein A affinity chromatography(Pharmacia).

To assess whether hamster anti-mouse LT-α and LT-β mAbs could block LTligand binding to mLT-β-R, we used TIMI-4 cells (ATCC), a murine T cellline that expresses surface LT ligand following PMA activation for 7hours. Hamster anti-mLT-α or anti-mLT-β mAbs were preincubated with thecells for 30 minutes at 4° C. and then washed twice. The washed cellswere incubated with 1 μg/ml of mLT-β-R-Fc at 4° C. After 30 minutes,cells were washed free of unbound mLT-β-R-Fc and then incubated for 30minutes with 10 μg/ml of phycoerythrin-labelled donkey anti-human IgG todetect bound mLT-β-R-Fc. The amount of bound fluorescent label wasdetermined by FACS analysis and the mean fluorescence intensity wascalculated.

Using this analysis, it was found that the hamster anti-mLT-β mAb couldeffectively block soluble LT-β receptor binding to T cell surface LTligand. The results are shown in Table 2.

                  TABLE 2    ______________________________________    Ability of Anti-mouse LT-β Monoclonal Antibody To    Inhibit mLT-β-R-Fc Binding To Murine Surface LT Ligand               Anti-mLT-β                           Anti-mLT-α               (BB.F6)     (AF.B3)    Conc. mAb (μg/ml)                 MFCI.sup.b                         % Inh.sup.c                                   MFCI.sup.b                                         % Inh.sup.c    ______________________________________    0.sup.a      6       --        6     --    0            85       0        85    0     0.01        71      18        84    2     0.03        67      23        86    0    0.1          51      44        86    0    0.3          36      62        84    2    1.0          29      71        89    0    3.0          17      86        88    0    10.0         11      94        95    0    30.0         10      95        94    0    100.0        8       98        92    0    ______________________________________     .sup.a no receptor added     .sup.b Mean Fluorescence Channel No.     .sup.c Percent Inhibition

EXAMPLE 8 LT-β-R Blocking Agents Inhibit Th1-Mediated ContactHypersensitivity in Mouse

20 g female Balb/c mice (Jackson Laboratories, Bar Harbor, Me.) wereinitially sensitized by applying 25 μL of 0.5% 2,4-dinitrofluorobenzene(DNFB) in 4:1 v/v acetone:olive oil onto the bottom of each hind foot.Twenty-four hours after the initial sensitization, we again sensitizedeach mouse with 25 μl of the same solution. Sensitizations wereperformed while restraining the unanesthetized mouse. On day 5 (120hours after the initial sensitization), we anesthetized the mice with90:10 mg/kg ketamine:xylazine (i.p.) and applied a sub-irritant dose of10 μl of 0.2% DNFB to the dorsal and ventral surfaces of the left ear.The right ear received a similar application of the 4:1 v/vacetone:olive oil vehicle.

Four hours after challenging the immune response, we administeredincreasing concentrations of the mLT-β-R-Fc (0.08-5.0 mg/kg; Example 2)to the mice in 0.1 ml of phosphate buffered saline (PBS) by intravenousinjection. Injections of PBS buffer alone, or 20 mg/kg of a human IgGfusion protein (LFA3-Fc) (Miller et al., J. Exp. Med., 178, pp. 211-22(1993)) served as negative controls. Injection of 8 mg/kg of ananti-VLA4-specific mAb (PS/2 mAb; Chisolm et al., Eur. J. Immunol., 23,pp. 682-88 (1993))--which is known to inhibit CHS by blocking the influxof T cells into the challenge site--served as a positive control. Groupsof four to eight mice were treated per concentration of antibody.

Twenty four hours after challenge, mice were again anesthetized withketamine:xylazine and the ear thickness of both ears measured with anengineer's micrometer to an accuracy of 10⁻⁴ inches. The ear swellingresponse for each mouse was the difference between its control- andDNFB-challenged ear thickness.

Typical uninhibited ear swelling responses were 95-110×10⁻⁴ inches.Inhibition of the ear swelling response was judged by comparison oftreated groups with their negative control group. Statisticalsignificance of the difference among treatment groups was evaluatedusing one-way analysis of variance followed by computation of theTukey-Kramer Honestly Significant Difference (JMP, SAS Institute) usingp<0.05.

FIG. 5 shows that administering increasing concentrations of mLT-β-R-Fccauses a significant reduction in the ear swelling response ofDNFB-treated mice compared to uninhibited DNFB-treated control animals(PBS and LFA3-Fc). Soluble LT-β-R (from about 1-5 mg/kg) can block thiscontact DTH reaction as effectively as the inhibitor anti-VLA4-specificmAb. The portion of this ear swelling assay which is not inhibitedprobably results from "nonspecific" granulocyte infiltration.

EXAMPLE 9 Murine LT-β-R-Fc Does Not Affect a Primary ImmunoglobulinResponse to a Nominal Antigen in Mouse

Female Balb/c mice, 2-3 months old (Jackson Laboratories, Bar Harbor,Me.), were injected with either 100-200 μg of mLT-β-R-Fc or LFA3-Fc (acontrol IgG1 fusion protein) five times over the course of a ten dayperiod. Another group of control mice was left untreated.

After the second injection, all mice were injected in the base of thetail with 100 μl of complete Freund's adjuvant containing 100 μg ofemulsified ovalbumin. At day 11, the mice were bled and serum prepared.The mice were sacrificed and the draining lymph nodes and spleenscollected. Primary serum anti-ovalbumin-specific antibody titers wereanalyzed using an ELISA specific for each IgG1 , IgG2a and IgM isotype.

Microtiter plates were coated with ovalbumin, blocked, and variousdilutions of the serum applied. Bound antibody was detected withanti-IgG1, -IgG2a or -IgM-specific reagents. The reciprocal of thedilution required to decrease the binding to 50% of the saturated levelwas defined as the titer.

FIG. 6 shows that none of the treatments affected the primary antibodytiter following ovalbumin immunization. By comparison, interfering withCD40 ligand-induced CD40 receptor signalling completely blocks theantigen-specific IgG response in mice (Renshaw et al., J. Exp. Med.,180, pp. 1889-1900 (1994)). CD40 is another ligand/receptor pair in theTNF family.

These experiments indicate that a therapy based on the inhibition ofLT-receptor interactions with its ligand(s) will not affect the antibodyproduction aspects of an immune response. Moreover, the normal antibodyresponse also indicates that an intensive treatment of mice with solublemLT-β-R-Fc was not toxic, further indicating the useful therapeuticnature of the compositions and methods set forth in this invention.

EXAMPLE 10 Effects of In Vivo mLT-β-R-Fc Treatment on Ex VivoProliferation Response of Primary Lymph Node Lymphocytes

To examine further the effect of an LT-β-R blocking agent on a Th2cell-dependent response in vivo, animals were treated with mLT-β-R-Fcand the ability of their lymphocytes to respond to antigen was measured.Mice were immunized with 100 μg of ovalbumin in complete Freund'sadjuvant in the tailbase on day 2. Mice were injected with 200 μg/mouseof mLT-β-R-Fc or LFA3-Fc i.p. in 250 μl of PBS on days 1, 2, 4, 7 and 9.On day 11, lymph nodes were isolated, a single cell suspension preparedand cells were resuspended in RPMI 1640 medium with 10% FCS, glutamineand Penicillin/Streptomycin.

A 0.1 ml aliquot of 10⁵ cells was added to each well of a 96-well platecontaining 0.1 ml RPMI with increasing concentrations of ovalbumin. Theplate was incubated for 3 days at 37° C. and then pulsed for 18 hourswith 1 μCi/well 3H-thymidine (New England Nuclear). The cells on eachplate were harvested onto glass fiber filter paper using a TomTecharvester (TomTec) and the incorporated thymidine determined by liquidscintillation counting using a LKB Wallac Beta Plate Counter (PharmaciaLKB Nuclear, Gaithersburg, Md.).

FIG. 7 shows that extensive in vivo treatment with mLT-β-R-Fc did notaffect the subsequent ability of ovalbumin-primed lymph node lymphocytesto proliferate in response to ex vivo restimulation with the ovalbuminantigen. Parallel treatment with LFA3-Fc, another human IgG-Fc fusionprotein, serves as a negative control.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 1    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 197 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    - Ser Gln Pro Gln Ala Val Pro Pro Tyr Ala Se - #r Glu Asn Gln Thr Cys    #                15    - Arg Asp Gln Glu Lys Glu Tyr Tyr Glu Pro Gl - #n His Arg Ile Cys Cys    #            30    - Ser Arg Cys Pro Pro Gly Thr Tyr Val Ser Al - #a Lys Cys Ser Arg Ile    #        45    - Arg Asp Thr Val Cys Ala Thr Cys Ala Glu As - #n Ser Tyr Asn Glu His    #    60    - Trp Asn Tyr Leu Thr Ile Cys Gln Leu Cys Ar - #g Pro Cys Asp Pro Val    #80    - Met Gly Leu Glu Glu Ile Ala Pro Cys Thr Se - #r Lys Arg Lys Thr Gln    #                95    - Cys Arg Cys Gln Pro Gly Met Phe Cys Ala Al - #a Trp Ala Leu Glu Cys    #           110    - Thr His Cys Glu Leu Leu Ser Asp Cys Pro Pr - #o Gly Thr Glu Ala Glu    #       125    - Leu Lys Asp Glu Val Gly Lys Gly Asn Asn Hi - #s Cys Val Pro Cys Lys    #   140    - Ala Gly His Phe Gln Asn Thr Ser Ser Pro Se - #r Ala Arg Cys Gln Pro    145                 1 - #50                 1 - #55                 1 -    #60    - His Thr Arg Cys Glu Asn Gln Gly Leu Val Gl - #u Ala Ala Pro Gly Thr    #               175    - Ala Gln Ser Asp Thr Thr Cys Lys Asn Pro Le - #u Glu Pro Leu Pro Pro    #           190    - Glu Met Ser Gly Thr            195    __________________________________________________________________________

What is claimed is:
 1. A method for altering a delayed typehypersensitivity response in an animal comprising the step ofadministering a pharmaceutical composition which comprises atherapeutically effective amount of a lymphotoxin -β receptor blockingagent and a pharmaceutically acceptable carrier.
 2. The method accordingto claim 1, wherein the lymphotoxin-β receptor blocking agent isselected from the group consisting of a soluble lymphotoxin-β receptorcomprising a functional sequence of amino acids selected from the aminoacids of SEQ.ID.NO.1, an antibody directed against lymphotoxin-βreceptor, and an antibody directed against a surface LT ligandcomprising at least one lymphotoxin-β subunit.
 3. The method accordingto claim 2, wherein the animal is a mammal.
 4. The method according toclaim 3, wherein the mammal is a human.
 5. The method according to claim1, wherein the lymphotoxin-β-receptor blocking agent comprises a solublelymphotoxin-β receptor comprising a functional sequence of amino acidsselected from the amino acids of SEQ.ID.NO.1, and having a ligandbinding domain that can bind to a surface LT ligand comprising at leastone lymphotoxin-β subunit.
 6. The method according to claim 5, whereinthe soluble lymphotoxin-β receptor further comprises a humanimmunoglobulin Fc domain.
 7. The method according to claim 1, whereinthe LT-β-R blocking agent comprises a monoclonal antibody directedagainst LT-β receptor.
 8. The method according to claim 7, wherein thecomposition is administered in an amount sufficient to coat LT-βreceptor-positive cells for 1 to 14 days.
 9. The method according toclaim 4, wherein the LT-β-R blocking agent comprises anti-human LT-β-RmAb BDA8 produced by the hybridoma cell line BD.A8.AB9 (ATCC AccessionNo: HB11798).
 10. The method according to claim 1, wherein the LT-β-Rblocking agent comprises a monoclonal antibody directed against surfaceLT ligand.
 11. The method according to claim 10, wherein the compositionis administered in an amount sufficient to coat surface LTligand-positive cells for 1 to 14 days.
 12. The method according toclaim 10, wherein the antibody is directed against a subunit of the LTligand.
 13. The method according to claim 4, wherein the LT-β-R blockingagent comprises anti-human LT-β mAb B9 produced by the hybridoma cellline B9.C9.1 (ATCC Accession No: 11962).
 14. The method according toclaim 3, wherein the mammal is a mouse and the LT-β-R blocking agentcomprises a monoclonal antibody directed against a murine surface LTligand.
 15. A method for treating inflammatory bowel disease in ananimal comprising the step of administering a pharmaceutical compositionwhich comprises a therapeutically effective amount of a lymphotoxin-βreceptor blocking agent and a pharmaceutically acceptable carrier. 16.The method according to claim 15 wherein the lymphotoxin-β receptorblocking agent is selected from the group consisting of a solublelymphotoxin-β receptor comprising a functional sequence of amino acidsselected from the amino acids of SEQ. ID.NO.1, an antibody directedagainst lymphotoxin β receptor, and an antibody directed against asurface LT ligand comprising at least one lymphotoxin-β subunit.